CN111334476A - Application of HSD17B1 gene and/or NUCB2 gene and regulation method of granulosa cells - Google Patents
Application of HSD17B1 gene and/or NUCB2 gene and regulation method of granulosa cells Download PDFInfo
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- CN111334476A CN111334476A CN202010177696.0A CN202010177696A CN111334476A CN 111334476 A CN111334476 A CN 111334476A CN 202010177696 A CN202010177696 A CN 202010177696A CN 111334476 A CN111334476 A CN 111334476A
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Abstract
本发明提供了一种HSD17B1基因和/或NUCB2基因的应用和颗粒细胞的调控方法，涉及生物技术领域。该颗粒细胞的调控方法包括调控颗粒细胞中HSD17B1基因和/或NUCB2基因的表达；该颗粒细胞的调控方法至少可以实现如下其中一种调控：调控颗粒细胞凋亡，调控颗粒细胞增殖，调控颗粒细胞分泌卵泡发育相关生殖激素，和，调控颗粒细胞中PI3K‑Akt信号通路；其中卵泡发育相关生殖激素包括E2、P4、ACT、INH和FS中的至少一种。
The invention provides an application of HSD17B1 gene and/or NUCB2 gene and a method for regulating granulosa cells, and relates to the field of biotechnology. The method for regulating granulosa cells includes regulating the expression of HSD17B1 gene and/or NUCB2 gene in granulosa cells; the method for regulating granulosa cells can achieve at least one of the following: regulating granulosa cell apoptosis, regulating granulosa cell proliferation, regulating granulosa cells Secretion of reproductive hormones related to follicle development, and regulation of PI3K-Akt signaling pathway in granulosa cells; wherein reproductive hormones related to follicle development include at least one of E 2 , P 4 , ACT, INH and FS.
Description
技术领域technical field
本发明涉及生物技术领域，尤其是涉及一种HSD17B1基因和/或NUCB2基因的应用和颗粒细胞的调控方法。The present invention relates to the field of biotechnology, in particular to an application of HSD17B1 gene and/or NUCB2 gene and a method for regulating granulosa cells.
背景技术Background technique
卵泡是哺乳动物生殖系统的核心功能单位，卵泡发育涉及神经调节、神经内分泌调节、内分泌调节以及旁分泌调节和自然分泌等一系列协调生理过程。卵泡由膜细胞、颗粒细胞和卵母细胞组成。在卵巢中，卵母细胞发育成熟的过程是在由颗粒细胞包绕的环境中完成的，临近卵母细胞的颗粒细胞具有较长的细胞质延伸，渗透到透明带中与卵母细胞膜形成间隙连接，因此颗粒细胞可以为卵母细胞的发育和成熟提供必需的小分子。颗粒细胞为卵母细胞的发育提供了充足的物理和化学条件，对支持卵母细胞发育成熟、传递内分泌即其他环境信号由至关重要的作用。因此开发调控颗粒细胞的调控方法以及产品，对了解颗粒细胞以及卵泡生理生化功能具有重要作用。The follicle is the core functional unit of the mammalian reproductive system, and follicle development involves a series of coordinated physiological processes such as neuromodulation, neuroendocrine regulation, endocrine regulation, paracrine regulation and natural secretion. The follicle consists of theca cells, granulosa cells and oocytes. In the ovary, the process of oocyte maturation is completed in an environment surrounded by granulosa cells. The granulosa cells adjacent to the oocyte have longer cytoplasmic extensions that penetrate into the zona pellucida to form gap junctions with the oocyte membrane , so granulosa cells can provide essential small molecules for oocyte development and maturation. Granulosa cells provide sufficient physical and chemical conditions for the development of oocytes, and play a crucial role in supporting the maturation of oocytes and transmitting endocrine and other environmental signals. Therefore, the development of regulatory methods and products for regulating granulosa cells plays an important role in understanding the physiological and biochemical functions of granulosa cells and follicles.
有鉴于此，特提出本发明。In view of this, the present invention is proposed.
发明内容SUMMARY OF THE INVENTION
本发明的第一目的在于提供一种卵巢颗粒细胞的调控方法，该调控方法至少能够调控颗粒细胞凋亡和/或调控颗粒细胞分泌卵泡发育相关生殖激素。The first object of the present invention is to provide a method for regulating ovarian granulosa cells, which can at least regulate the apoptosis of granulosa cells and/or regulate the secretion of reproductive hormones related to follicle development by granulosa cells.
本发明的第二目的在于提供HSD17B1基因和/或NUCB2基因在制备用于调控颗粒细胞的产品中的应用。The second object of the present invention is to provide the application of HSD17B1 gene and/or NUCB2 gene in the preparation of a product for regulating granulosa cells.
为解决上述技术问题，本发明特采用如下技术方案：In order to solve the above-mentioned technical problems, the present invention adopts the following technical solutions:
根据本发明的一个方面，本发明提供了一种颗粒细胞的调控方法，包括调控颗粒细胞中HSD17B1基因和/或NUCB2基因的表达；According to one aspect of the present invention, the present invention provides a method for regulating granulosa cells, comprising regulating the expression of HSD17B1 gene and/or NUCB2 gene in granulosa cells;
对颗粒细胞的调控包括：调控颗粒细胞凋亡，调控颗粒细胞增殖，调控颗粒细胞分泌卵泡发育相关生殖激素，和，调控颗粒细胞中PI3K-Akt信号通路中的至少一种；所述卵泡发育相关生殖激素包括E2、P4、ACT、INH和FS中的至少一种。The regulation of granulosa cells includes: regulation of granulosa cell apoptosis, regulation of granulosa cell proliferation, regulation of granulosa cells to secrete follicle development-related reproductive hormones, and regulation of at least one of PI3K-Akt signaling pathways in granulosa cells; the follicle development-related Reproductive hormones include at least one of E2 , P4 , ACT, INH, and FS.
根据本发明的另一个方面，本发明还提供了HSD17B1基因和/或NUCB2基因在制备用于调控颗粒细胞的产品中的应用；所述调控颗粒细胞包括：调控颗粒细胞凋亡，调控颗粒细胞增殖，调控颗粒细胞分泌卵泡发育相关生殖激素，和，调控颗粒细胞中p-AKT的表达中的至少一种；所述卵泡发育相关生殖激素包括E2、P4、ACT、INH和FS中的至少一种。According to another aspect of the present invention, the present invention also provides the use of HSD17B1 gene and/or NUCB2 gene in the preparation of a product for regulating granulosa cells; the regulating granulosa cells includes: regulating granulosa cell apoptosis, regulating granulosa cell proliferation , regulating granulosa cells to secrete follicle development-related reproductive hormones, and, regulating at least one of p-AKT expression in granulosa cells; the follicular development-related reproductive hormones include at least one of E 2 , P 4 , ACT, INH and FS A sort of.
根据本发明的另一个方面，本发明还提供了一种用于调控颗粒细胞的产品，包括如下(x1)～(x4)中的至少一种；According to another aspect of the present invention, the present invention also provides a product for regulating granulosa cells, comprising at least one of the following (x1) to (x4);
(x1)用于过表达HSD17B1基因的物质；(x1) Substances for overexpressing the HSD17B1 gene;
(x2)用于抑制HSD17B1基因表达的物质；(x2) Substances for inhibiting HSD17B1 gene expression;
(x3)用于过表达NUCB2基因的物质；(x3) Substances for overexpressing the NUCB2 gene;
(x4)用于抑制NUCB2基因表达的物质。(x4) Substances for inhibiting NUCB2 gene expression.
根据本发明的另一个方面，本发明还提供了所述颗粒细胞的调控方法、或所述产品在调控卵泡和/或卵巢发育或制备调控卵泡和/或卵巢发育的产品中的应用。According to another aspect of the present invention, the present invention also provides the method for regulating granulosa cells, or the application of the product in regulating follicle and/or ovary development or preparing a product for regulating follicle and/or ovary development.
与现有技术相比，本发明具有如下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
本发明通过实验发现：(1)HSD17B1基因和NUCB2基因与颗粒细胞的凋亡相关，HSD17B1基因和NUCB2基因均分别独立的具有促进颗粒细胞凋亡的作用，并且下调NUCB2基因表达还具有促进颗粒细胞增殖的作用。(2)HSD17B1基因和NUCB2基因与颗粒细胞分泌卵泡发育相关生殖激素的水平相关，HSD17B1基因具有促进E2、P4和ACT分泌，以及抑制INH和FS分泌的作用；NUCB2基因具有抑制E2、P4和ACT分泌，以及促进INH和FS分泌的作用。(3)NUCB2基因的表达和PI3K-Akt(磷脂酰肌醇-3激酶/蛋白激酶B)信号通路相关，NUCB2基因的表达能够抑制PI3K-Akt信号通路。The present invention finds through experiments that: (1) HSD17B1 gene and NUCB2 gene are related to the apoptosis of granulosa cells. Both HSD17B1 gene and NUCB2 gene independently have the effect of promoting the apoptosis of granulosa cells, and down-regulating the expression of NUCB2 gene can also promote the apoptosis of granulosa cells. effect of proliferation. (2) HSD17B1 gene and NUCB2 gene are related to the levels of reproductive hormones secreted by granulosa cells related to follicle development. HSD17B1 gene can promote the secretion of E 2 , P 4 and ACT, and inhibit the secretion of INH and FS; NUCB2 gene can inhibit the secretion of E 2 , P 4 and ACT. P4 and ACT secretion, and the role of promoting INH and FS secretion. (3) The expression of NUCB2 gene is related to the PI3K-Akt (phosphatidylinositol-3 kinase/protein kinase B) signaling pathway, and the expression of NUCB2 gene can inhibit the PI3K-Akt signaling pathway.
基于本发明实验中发现的HSD17B1基因和/或NUCB2基因对颗粒细胞的调控作用，本发明提供了颗粒细胞的调控方法和HSD17B1基因和/或NUCB2基因在制备用于调控颗粒细胞的产品中的应用。本发明提供的颗粒细胞的调控方法和产品有助于进一步了解卵泡发育后期卵母细胞成熟机制，为完善卵泡发育相关信号通路，揭示卵泡发育的机制以及筛选卵泡发育相关因子的工作提供了技术和物质保障；对优化卵母细胞体外培养体系，了解并调控哺乳动物的超数排卵，胚胎体外生产以及多胎品种选育等具有重要意义；有助于养殖业中牲畜的选育和繁殖工作的进行。Based on the regulation effect of HSD17B1 gene and/or NUCB2 gene on granulosa cells found in the experiments of the present invention, the present invention provides a regulation method of granulosa cells and the application of HSD17B1 gene and/or NUCB2 gene in the preparation of products for regulating granulosa cells . The method and product for regulating granulosa cells provided by the present invention help to further understand the oocyte maturation mechanism in the later stage of follicle development, and provide technical and technical support for improving follicle development-related signaling pathways, revealing the follicle development mechanism and screening follicle development-related factors. Material guarantee; it is of great significance for optimizing the oocyte culture system in vitro, understanding and regulating mammalian superovulation, in vitro embryo production, and breeding of multiple littermates; it is helpful for the selection and breeding of livestock in the breeding industry. .
附图说明Description of drawings
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to illustrate the specific embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.
图1为本发明实施例1中转染pIRES2-ZsGreen1-HSD17B1后卵巢颗粒细胞中HSD17B1相对表达量；Figure 1 shows the relative expression of HSD17B1 in ovarian granulosa cells after transfection of pIRES2-ZsGreen1-HSD17B1 in Example 1 of the present invention;
图2为本发明实施例1中siRNA-3对HSD17B1基因表达的干扰效果；Figure 2 is the interference effect of siRNA-3 on HSD17B1 gene expression in Example 1 of the present invention;
图3为本发明实施例2中HSD17B1基因干扰及过表达后流式细胞凋亡检测结果；Figure 3 is the result of flow cytometry apoptosis detection after HSD17B1 gene interference and overexpression in Example 2 of the present invention;
图4为本发明实施例2中HSD17B1基因干扰及过表达后颗粒细胞凋亡率；Figure 4 shows the apoptosis rate of granulosa cells after HSD17B1 gene interference and overexpression in Example 2 of the present invention;
图5为本发明实施例2中HSD17B1基因干扰及过表达后颗粒细胞增殖率；Figure 5 is the proliferation rate of granulosa cells after HSD17B1 gene interference and overexpression in Example 2 of the present invention;
图6为本发明实施例3中HSD17B1基因对绵羊颗粒细胞分泌E2的影响；Figure 6 is the effect of HSD17B1 gene on E2 secretion by sheep granulosa cells in Example 3 of the present invention;
图7为本发明实施例3中HSD17B1基因对绵羊颗粒细胞分泌P4的影响；Figure 7 is the effect of HSD17B1 gene on the secretion of P4 by sheep granulosa cells in Example 3 of the present invention;
图8为本发明实施例3中HSD17B1基因对绵羊颗粒细胞分泌ACT的影响；Figure 8 is the effect of HSD17B1 gene on the secretion of ACT by sheep granulosa cells in Example 3 of the present invention;
图9为本发明实施例3中HSD17B1基因对绵羊颗粒细胞分泌INH的影响；Figure 9 is the effect of HSD17B1 gene on the secretion of INH by sheep granulosa cells in Example 3 of the present invention;
图10为本发明实施例3中HSD17B1基因对绵羊颗粒细胞分泌FS的影响；Figure 10 shows the effect of HSD17B1 gene on the secretion of FS by sheep granulosa cells in Example 3 of the present invention;
图11为本发明实施例4中转染pIRES2-ZsGreen1-NUCB2后卵巢颗粒细胞中NUCB2相对表达量；Figure 11 shows the relative expression of NUCB2 in ovarian granulosa cells after transfection of pIRES2-ZsGreen1-NUCB2 in Example 4 of the present invention;
图12本发明实施例5中NUCB2基因干扰及过表达后流式细胞凋亡检测结果；Figure 12. Flow cytometric apoptosis detection results after NUCB2 gene interference and overexpression in Example 5 of the present invention;
图13本发明实施例5中NUCB2基因干扰及过表达后颗粒细胞凋亡率；Figure 13 Apoptosis rate of granulosa cells after NUCB2 gene interference and overexpression in Example 5 of the present invention;
图14本发明实施例5中NUCB2基因干扰及过表达后颗粒细胞增殖率；Figure 14. The proliferation rate of granulosa cells after NUCB2 gene interference and overexpression in Example 5 of the present invention;
图15为发明实施例6中Western Blot检测NUCB2基因过表达和干扰处理后p-AKT蛋白的表达水平；Figure 15 shows the expression level of p-AKT protein after NUCB2 gene overexpression and interference treatment detected by Western Blot in Example 6 of the invention;
图16为发明实施例6中Western Blot检测NUCB2基因过表达和干扰处理后p-AKT蛋白的免疫印迹图；Figure 16 is an immunoblot of p-AKT protein after Western Blot detection of NUCB2 gene overexpression and interference treatment in Example 6 of the invention;
图17为本发明实施例3中NUCB2基因对绵羊颗粒细胞分泌E2的影响；Figure 17 shows the effect of NUCB2 gene on E2 secretion by sheep granulosa cells in Example 3 of the present invention;
图18为本发明实施例3中NUCB2基因对绵羊颗粒细胞分泌P4的影响；Figure 18 is the effect of NUCB2 gene on the secretion of P4 by sheep granulosa cells in Example 3 of the present invention;
图19为本发明实施例3中NUCB2基因对绵羊颗粒细胞分泌ACT的影响；Figure 19 shows the effect of NUCB2 gene on the secretion of ACT by sheep granulosa cells in Example 3 of the present invention;
图20为本发明实施例3中NUCB2基因对绵羊颗粒细胞分泌INH的影响；Figure 20 is the effect of NUCB2 gene on the secretion of INH by sheep granulosa cells in Example 3 of the present invention;
图21为本发明实施例3中NUCB2基因对绵羊颗粒细胞分泌FS的影响。Figure 21 shows the effect of NUCB2 gene on the secretion of FS by sheep granulosa cells in Example 3 of the present invention.
具体实施方式Detailed ways
下面将结合实施例对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
根据本发明的一个方面，本发明提供了一种颗粒细胞的调控方法，包括调控颗粒细胞中HSD17B1基因和/或NUCB2基因的表达。HSD17B1基因编码17β-羟类固醇脱氢酶1(17beta-hydroxysteroid dehydrogenases，HSD17B1)，其具有雌激素激活和雄激素失活的双重功能，是催化雌激素激活的最后一步。NUCB2基因编码的核组蛋白2可以水解为Nesfatin-1(1-82位氨基酸)，Nesfatin-1在糖代谢、神经调节方面均可发挥作用。According to one aspect of the present invention, the present invention provides a method for regulating granulosa cells, comprising regulating the expression of HSD17B1 gene and/or NUCB2 gene in granulosa cells. The HSD17B1 gene encodes 17beta-hydroxysteroid dehydrogenase 1 (HSD17B1), which has dual functions of estrogen activation and androgen inactivation, and is the last step in catalyzing estrogen activation. The
本发明对颗粒细胞的调控包括如下几个方面：调控颗粒细胞凋亡，调控颗粒细胞增殖，调控颗粒细胞分泌卵泡发育相关生殖激素，以及，调控颗粒细胞中PI3K-Akt信号通路。The regulation of granulosa cells in the present invention includes the following aspects: regulation of apoptosis of granulosa cells, regulation of proliferation of granulosa cells, regulation of granulosa cells to secrete reproductive hormones related to follicle development, and regulation of PI3K-Akt signaling pathway in granulosa cells.
本发明通过实验发现，HSD17B1基因和NUCB2基因与颗粒细胞的凋亡相关，上调HSD17B1基因和NUCB2基因的表达，均分别独立的具有促进颗粒细胞凋亡的作用；同时下调HSD17B1基因和NUCB2基因的表达，均分别独立的具有抑制颗粒细胞凋亡的作用。其中下调NUCB2基因表达还具有促进颗粒细胞增殖的作用，但上调NUCB2基因表达以及调控HSD17B1基因的表达均无法调控颗粒细胞的增殖。The present invention finds through experiments that HSD17B1 gene and NUCB2 gene are related to the apoptosis of granulosa cells, up-regulating the expression of HSD17B1 gene and NUCB2 gene, both independently have the effect of promoting apoptosis of granulosa cells; meanwhile, down-regulating the expression of HSD17B1 gene and NUCB2 gene , all independently have the effect of inhibiting apoptosis of granulosa cells. Among them, down-regulation of NUCB2 gene expression can also promote the proliferation of granulosa cells, but up-regulation of NUCB2 gene expression and regulation of HSD17B1 gene expression cannot regulate the proliferation of granulosa cells.
本发明通过实验发现，HSD17B1基因和NUCB2基因与颗粒细胞分泌卵泡发育相关生殖激素的水平相关。其中卵泡发育相关生殖激素包括雌二醇(E2)、孕酮(P4)、激活素(ACT)、抑制素(INH)和卵泡抑素(FS)中的至少一种。上调HSD17B1基因表达和下调NUCB2基因表达分别独立的具有促进E2、P4和ACT中至少一种的分泌的作用，以及抑制INH和FS中至少一种的分泌的作用。下调HSD17B1基因表达和上调NUCB2基因表达分别独立的具有抑制E2、P4和ACT中至少一种的分泌的作用，以及促进INH和FS中至少一种的分泌的作用。The present invention finds through experiments that the HSD17B1 gene and the NUCB2 gene are related to the levels of reproductive hormones secreted by granulosa cells related to follicle development. The reproductive hormones related to follicle development include at least one of estradiol (E 2 ), progesterone (P 4 ), activin (ACT), inhibin (INH) and follistatin (FS). Up-regulation of HSD17B1 gene expression and down-regulation of NUCB2 gene expression independently have the effect of promoting the secretion of at least one of E 2 , P 4 and ACT, and inhibiting the secretion of at least one of INH and FS, respectively. Down-regulation of HSD17B1 gene expression and up-regulation of NUCB2 gene expression independently inhibited the secretion of at least one of E 2 , P 4 and ACT, and promoted the secretion of at least one of INH and FS, respectively.
本发明通过实验还发现，NUCB2基因的表达和PI3K-Akt(磷脂酰肌醇-3激酶/蛋白激酶B)信号通路相关，上调NUCB2基因表达，PI3K-Akt的标志蛋白p-AKT表现为低表达，下调NUCB2基因表达则p-AKT表现为高表达，说明上调NUCB2基因的表达抑制了PI3K-Akt信号通路，而下调NUCB2基因的表达可以活化PI3K-Akt信号通路。The present invention also finds through experiments that the expression of NUCB2 gene is related to PI3K-Akt (phosphatidylinositol-3 kinase/protein kinase B) signaling pathway, the expression of NUCB2 gene is up-regulated, and p-AKT, the marker protein of PI3K-Akt, shows low expression , down-regulation of NUCB2 gene expression resulted in high expression of p-AKT, indicating that up-regulation of NUCB2 gene expression inhibited the PI3K-Akt signaling pathway, while down-regulation of NUCB2 gene expression could activate the PI3K-Akt signaling pathway.
基于本发明发现的HSD17B1基因和NUCB2基因对颗粒细胞的调控作用，本发明提供的颗粒细胞的调控方法至少可以采用如下实施方式中的一种或多种实现：Based on the regulatory effect of HSD17B1 gene and NUCB2 gene on granulosa cells found in the present invention, the regulation method of granulosa cells provided by the present invention can be realized by at least one or more of the following embodiments:
在一些可选的实施方式中，上调HSD17B1基因表达，以实现如下调控作用中的至少一种：促进颗粒细胞凋亡；促进E2、P4和ACT中至少一种的分泌；以及，抑制INH和FS中至少一种的分泌。In some optional embodiments, HSD17B1 gene expression is up-regulated to achieve at least one of the following regulatory effects: promoting granulosa cell apoptosis; promoting secretion of at least one of E 2 , P 4 and ACT; and inhibiting INH and secretion of at least one of FS.
在一些可选的实施方式中，下调HSD17B1基因表达，以实现如下调控作用中的至少一种：抑制颗粒细胞凋亡；抑制E2、P4和ACT中至少一种的分泌；以及，促进INH和FS中至少一种的分泌。In some alternative embodiments, the expression of HSD17B1 gene is down-regulated to achieve at least one of the following regulatory effects: inhibiting granulosa cell apoptosis; inhibiting the secretion of at least one of E2 , P4 and ACT ; and, promoting INH and secretion of at least one of FS.
在一些可选的实施方式中，上调NUCB2基因表达，以实现如下调控作用中的至少一种：促进颗粒细胞凋亡；抑制E2、P4和ACT中至少一种的分泌；促进INH和FS中至少一种的分泌；以及，抑制颗粒细胞中PI3K-Akt信号通路。In some optional embodiments, the expression of NUCB2 gene is up-regulated to achieve at least one of the following regulatory effects: promoting granulosa cell apoptosis; inhibiting the secretion of at least one of E 2 , P 4 and ACT; promoting INH and FS The secretion of at least one of; and, inhibition of PI3K-Akt signaling pathway in granulosa cells.
在一些可选的实施方式中，下调NUCB2基因表达，以实现如下调控作用中的至少一种：抑制颗粒细胞凋亡；促进颗粒细胞增殖；促进E2、P4和ACT中至少一种的分泌；抑制INH和FS中至少一种的分泌；以及，活化颗粒细胞中PI3K-Akt信号通路。In some optional embodiments, NUCB2 gene expression is down-regulated to achieve at least one of the following regulatory effects: inhibiting granulosa cell apoptosis; promoting granulosa cell proliferation; promoting secretion of at least one of E 2 , P 4 and ACT ; inhibit the secretion of at least one of INH and FS; and, activate the PI3K-Akt signaling pathway in granulosa cells.
可以理解的是，所述颗粒细胞的调控方法可以采用上调HSD17B1基因表达、下调HSD17B1基因表达、上调NUCB2基因表达和下调NUCB2基因表达中的其中一种方式，也可以采用同时调控至少两种基因。例如在一些可选的实施方式中，同时上调HSD17B1基因和NUCB2基因的表达，以促进颗粒细胞凋亡；或，在另一些实施方式中，上调HSD17B1基因的表达，同时下调NUCB2基因表达，以实现促进E2、P4和ACT中至少一种的分泌，或者抑制INH和FS中至少一种的分泌。It can be understood that the regulation method of the granulosa cells can adopt one of the methods of up-regulating HSD17B1 gene expression, down-regulating HSD17B1 gene expression, up-regulating NUCB2 gene expression and down-regulating NUCB2 gene expression, or simultaneously regulating at least two genes. For example, in some optional embodiments, the expression of HSD17B1 gene and NUCB2 gene are up-regulated at the same time to promote apoptosis of granulosa cells; or, in other embodiments, the expression of HSD17B1 gene is up-regulated, and the expression of NUCB2 gene is down-regulated at the same time to achieve Promote the secretion of at least one of E2 , P4 and ACT, or inhibit the secretion of at least one of INH and FS.
需要说明的是，本发明不限制在调控颗粒细胞中HSD17B1基因和/或NUCB2基因表达的同时调控颗粒细胞中其他基因的表达，以实现对颗粒细胞中其他卵泡发育相关生殖激素、信号通路或者其他生理生化活动的调整。It should be noted that the present invention is not limited to regulating the expression of HSD17B1 gene and/or NUCB2 gene in granulosa cells while regulating the expression of other genes in granulosa cells, so as to realize the control of other reproductive hormones, signaling pathways or other related follicle development in granulosa cells. Adjustment of physiological and biochemical activities.
上述实施例中，促进或抑制E2、P4、ACT、INH和FS中至少一种的分泌，可以同时对这五种卵泡发育相关生殖激素进行调控，例如采用上调HSD17B1基因表达的方式，促进E2、P4和ACT的分泌，同时抑制INH和FS的分泌。在另外一些实施例中，也可以对E2、P4、ACT、INH和FS中的其中一种或几种调控，例如，采用上调HSD17B1基因表达的方式促进E2、P4和ACT的分泌，但同时调控其他基因，或添加能够抑制卵泡发育相关生殖激素的抑制剂，例如E2抑制剂和/或P4抑制剂，以实现对E2、P4、ACT、INH和FS中的其中一种或几种的选择调控。In the above embodiment, promoting or inhibiting the secretion of at least one of E 2 , P 4 , ACT, INH and FS can simultaneously regulate these five follicular development-related reproductive hormones, for example, by up-regulating the expression of the HSD17B1 gene to promote The secretion of E 2 , P 4 and ACT, while inhibiting the secretion of INH and FS. In other embodiments, one or more of E 2 , P 4 , ACT, INH and FS can also be regulated, for example, the secretion of E 2 , P 4 and ACT can be promoted by up-regulating the expression of HSD17B1 gene , but at the same time regulate other genes, or add inhibitors that can inhibit follicular development-related reproductive hormones, such as E 2 inhibitors and/or P 4 inhibitors, to achieve control of E 2 , P 4 , ACT, INH and FS among them One or more selective controls.
本发明不限制颗粒细胞的来源，颗粒细胞的来源包括但不限于牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂和人中的一种或多种。The present invention does not limit the source of granulosa cells, the sources of granulosa cells include but are not limited to cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink and humans one or more of.
在一些可选的实施方式中，调控的HSD17B1基因含有如SEQ ID NO.1所示序列；或，含有与SEQ ID NO.1所示序列至少90％以上同源性的序列。In some optional embodiments, the regulated HSD17B1 gene contains the sequence shown in SEQ ID NO.1; or, contains a sequence with at least 90% homology to the sequence shown in SEQ ID NO.1.
在一些可选的实施方式中，调控的NUCB2基因含有如SEQ ID NO.2所示序列；或，含有与SEQ ID NO.2所示序列至少90％以上同源性的序列。In some optional embodiments, the regulated NUCB2 gene contains the sequence shown in SEQ ID NO.2; or, contains a sequence with at least 90% homology to the sequence shown in SEQ ID NO.2.
其中90％以上同源性的序列指的是与目标序列由于核苷酸的缺失、插入或改变导致的不完全一致，但是至少具有90％相似度且与目标序列具有相同功能的序列。本文中其他部分的90％以上同源性的序列作同样的理解。A sequence with more than 90% homology refers to a sequence that is not completely identical to the target sequence due to nucleotide deletion, insertion or change, but has at least 90% similarity and has the same function as the target sequence. Sequences that are more than 90% homologous to other parts of the text are similarly understood.
上述实施方式中，“含有”指的是基因中除去含有如SEQ ID NO.1、SEQ ID NO.2或与他们同源的序列，还可以含有其他功能单元的编码序列，例如启动子、终止子、转录因子结合区域、增强子和载体序列中的至少一种；或者是所述基因具有如SEQ ID NO.1或SEQ IDNO.2所示序列或与他们同源的序列的cDNA。本文其他部分的基因“含有”如序列表中的所示序列也做相同的理解，不再赘述。In the above-mentioned embodiment, "contain" refers to the gene that removes the sequence that contains such as SEQ ID NO.1, SEQ ID NO.2 or homology with them, and can also contain coding sequences of other functional units, such as promoter, termination At least one of the sequences of promoter, transcription factor binding region, enhancer and vector; or the cDNA of the gene having the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 or a sequence homologous to them. The genes in other parts of this document "contain" the sequences shown in the sequence listing are also understood in the same way, and will not be repeated here.
上述SEQ ID NO.1所示序列和SEQ ID NO.2所示序列来源于绵羊，因此采用这两种序列的HSD17B1基因和NUCB2基因优选调控绵羊颗粒细胞。The sequence shown in SEQ ID NO. 1 and the sequence shown in SEQ ID NO. 2 are derived from sheep, so the HSD17B1 gene and NUCB2 gene using these two sequences are preferably used to regulate sheep granulosa cells.
调控绵羊颗粒细胞有助于进一步理解对多胎绵羊繁殖力产生影响的机制，可以加深对卵泡发育后期卵母细胞成熟机制的认识，进一步挖掘卵泡发育相关因子，完善卵泡发育相关信号通路，揭示卵泡发育的机制，优化绵羊卵母细胞体外培养体系。这对进一步开展绵羊超数排卵、羔羊超排、绵羊胚胎体外生产、多胎绵羊品种选育等具有重要意义。The regulation of granulosa cells is helpful to further understand the mechanism that affects the fertility of multiparity sheep. It can deepen the understanding of the oocyte maturation mechanism in the later stage of follicular development, further explore the factors related to follicle development, improve the signaling pathways related to follicle development, and reveal follicle development. mechanism to optimize the in vitro culture system of sheep oocytes. This is of great significance for further developing sheep superovulation, lamb superovulation, in vitro production of sheep embryos, and breeding of multiple litters.
根据本发明的另一个方面，本发明还提供了HSD17B1基因和/或NUCB2基因在制备用于调控颗粒细胞的产品中的应用。该应用和上述颗粒细胞的调控方法基于相同的发明构思，因此HSD17B1基因和/或NUCB2基因对颗粒细胞的调控机理不再赘述。According to another aspect of the present invention, the present invention also provides the use of HSD17B1 gene and/or NUCB2 gene in the preparation of a product for regulating granulosa cells. This application and the above-mentioned regulation method of granulosa cells are based on the same inventive concept, so the regulation mechanism of HSD17B1 gene and/or NUCB2 gene on granulosa cells will not be repeated.
用于调控颗粒细胞的产品至少采用如下实施方式中的一种或多种实现对颗粒细胞的调控作用：The product for regulating granulosa cells at least adopts one or more of the following embodiments to achieve the regulating effect on granulosa cells:
在一些可选的实施方式中，所述产品通过上调HSD17B1基因表达，以实现如下调控作用中的至少一种：促进颗粒细胞凋亡；促进E2、P4和ACT中至少一种的分泌；以及，抑制INH和FS中至少一种的分泌。In some optional embodiments, the product achieves at least one of the following regulatory effects by up-regulating HSD17B1 gene expression: promoting granulosa cell apoptosis; promoting the secretion of at least one of E 2 , P 4 and ACT; And, inhibit the secretion of at least one of INH and FS.
在一些可选的实施方式中，所述产品通过下调HSD17B1基因表达，以实现如下调控作用中的至少一种：抑制颗粒细胞凋亡；抑制E2、P4和ACT中至少一种的分泌；以及，促进INH和FS中至少一种的分泌。In some optional embodiments, the product can achieve at least one of the following regulatory effects by down-regulating HSD17B1 gene expression: inhibiting apoptosis of granulosa cells; inhibiting the secretion of at least one of E 2 , P 4 and ACT; And, promote the secretion of at least one of INH and FS.
在一些可选的实施方式中，所述产品通过上调NUCB2基因表达，以实现如下调控作用中的至少一种：促进颗粒细胞凋亡；抑制E2、P4和ACT中至少一种的分泌；促进INH和FS中至少一种的分泌；以及，抑制颗粒细胞中PI3K-Akt信号通路。In some optional embodiments, the product achieves at least one of the following regulatory effects by up-regulating NUCB2 gene expression: promoting granulosa cell apoptosis; inhibiting the secretion of at least one of E 2 , P 4 and ACT; Promote the secretion of at least one of INH and FS; and, inhibit the PI3K-Akt signaling pathway in granulosa cells.
在一些可选的实施方式中，所述产品通过下调NUCB2基因表达，以实现如下调控作用中的至少一种：抑制颗粒细胞凋亡；促进颗粒细胞增殖；促进E2、P4和ACT中至少一种的分泌；抑制INH和FS中至少一种的分泌；以及，活化颗粒细胞中PI3K-Akt信号通路。In some optional embodiments, the product can achieve at least one of the following regulatory effects by down-regulating NUCB2 gene expression: inhibiting granulosa cell apoptosis; promoting granulosa cell proliferation; promoting at least one of E 2 , P 4 and ACT secretion of one; inhibition of secretion of at least one of INH and FS; and, activation of the PI3K-Akt signaling pathway in granulosa cells.
可以理解的是，所述产品可以只上调HSD17B1基因表达、下调HSD17B1基因表达、上调NUCB2基因表达或下调NUCB2基因表达，也可以实现同时调控至少两种基因。例如在一些可选的实施方式中，所述产品同时下调HSD17B1基因和NUCB2基因的表达，以抑制颗粒细胞凋亡；或，在另一些实施方式中，下调HSD17B1基因的表达，同时上调NUCB2基因表达，以实现抑制E2、P4和ACT中至少一种的分泌，或者促进INH和FS中至少一种的分泌。需要说明的是，所述产品不限制在调控颗粒细胞中HSD17B1基因和/或NUCB2基因表达的同时还能够调控其他基因的表达，以实现对颗粒细胞中其他卵泡发育相关生殖激素、信号通路或者其他生理生化活动的调整。It can be understood that the product can only up-regulate HSD17B1 gene expression, down-regulate HSD17B1 gene expression, up-regulate NUCB2 gene expression or down-regulate NUCB2 gene expression, or can simultaneously regulate at least two genes. For example, in some optional embodiments, the product simultaneously down-regulates the expression of HSD17B1 gene and NUCB2 gene to inhibit apoptosis of granulosa cells; or, in other embodiments, down-regulates the expression of HSD17B1 gene and simultaneously up-regulates the expression of NUCB2 gene , so as to inhibit the secretion of at least one of E 2 , P 4 and ACT, or promote the secretion of at least one of INH and FS. It should be noted that the product is not limited to regulating the expression of HSD17B1 gene and/or NUCB2 gene in granulosa cells, but also can regulate the expression of other genes, so as to realize the control of other reproductive hormones, signaling pathways or other related follicle development in granulosa cells. Adjustment of physiological and biochemical activities.
上述实施例中，当所述产品用于促进或抑制E2、P4、ACT、INH和FS中至少一种的分泌，所述产品可以同时对这五种卵泡发育相关生殖激素进行调控，也可以只对其中的一种或部分卵泡发育相关生殖激素进行调控。例如所述产品中除去含有能够抑制NUCB2基因表达的物质，例如含有干扰NUCB2基因表达的siRNA，还含有能够促进或抑制卵泡发育相关生殖激素的药物，例如抑制ACT的制剂，以实现对E2和P4的促进作用以及INH和FS的抑制作用。In the above embodiment, when the product is used to promote or inhibit the secretion of at least one of E 2 , P 4 , ACT, INH and FS, the product can simultaneously regulate and control these five reproductive hormones related to follicular development, and also Only one or some of the reproductive hormones related to follicle development can be regulated. For example, the product removes substances that can inhibit NUCB2 gene expression, such as siRNA that interferes with NUCB2 gene expression, and also contains drugs that can promote or inhibit follicle development-related reproductive hormones, such as preparations that inhibit ACT, to achieve E2 and Promotion of P4 and inhibition of INH and FS.
本发明提供的用于调控颗粒细胞的产品，包括但不限于试剂、试剂盒、药物或培养基等。所述产品可以通过包含能够上调或下调基因的物质来实现对基因的调控。在一些实施例中，所述产品通过含有能够干扰基因表达的siRNA，或表达siRNA的DNA来下调基因的表达。在一些实施例中，所述产品通过含有表达HSD17B1基因和/或NUCB2基因的重组载体来实现基因的过表达，以达到上调基因表达的目的。在一些实施例中，所述产品还可以通过能够抑制靶基因表达的蛋白或化合物药物等来对基因进行调控。在一些实施例中，所述产品还可以通过含有能够表达具有调控基因表达作用的细胞或微生物来对基因进行调控。The products for regulating granulosa cells provided by the present invention include but are not limited to reagents, kits, medicines or culture media, and the like. The product can achieve gene regulation by including substances capable of up-regulating or down-regulating the gene. In some embodiments, the product downregulates gene expression by containing siRNA capable of interfering with gene expression, or siRNA expressing DNA. In some embodiments, the product achieves gene overexpression through a recombinant vector expressing HSD17B1 gene and/or NUCB2 gene, so as to achieve the purpose of up-regulating gene expression. In some embodiments, the product can also regulate genes through proteins or compound drugs that can inhibit the expression of target genes. In some embodiments, the product may also modulate genes by containing cells or microorganisms capable of expressing cells or microorganisms capable of modulating gene expression.
在一些可选的实施方式中，调控的HSD17B1基因含有如SEQ ID NO.1所示序列；或，含有与SEQ ID NO.1所示序列至少90％以上同源性的序列。In some optional embodiments, the regulated HSD17B1 gene contains the sequence shown in SEQ ID NO.1; or, contains a sequence with at least 90% homology to the sequence shown in SEQ ID NO.1.
在一些可选的实施方式中，调控的NUCB2基因含有如SEQ ID NO.2所示序列；或，含有与SEQ ID NO.2所示序列至少90％以上同源性的序列。In some optional embodiments, the regulated NUCB2 gene contains the sequence shown in SEQ ID NO.2; or, contains a sequence with at least 90% homology to the sequence shown in SEQ ID NO.2.
基于上述HSD17B1基因和/或NUCB2基因在制备用于调控颗粒细胞的产品中的应用的发明构思和基于本发明发现的HSD17B1基因和NUCB2基因对颗粒细胞的调控作用，本发明还提供了一种用于调控颗粒细胞的产品。所述用于调控颗粒细胞的产品包括(x1)用于过表达HSD17B1基因的物质；(x2)用于抑制HSD17B1基因表达的物质；(x3)用于过表达NUCB2基因的物质；和，(x4)用于抑制NUCB2基因表达的物质中的至少一种。Based on the inventive concept of the application of the above-mentioned HSD17B1 gene and/or NUCB2 gene in the preparation of products for regulating granulosa cells and the regulation effect of the HSD17B1 gene and NUCB2 gene on granulosa cells found in the present invention, the present invention also provides a method for using Products for the regulation of granulosa cells. The product for regulating granulosa cells includes (x1) a substance for overexpressing the HSD17B1 gene; (x2) a substance for inhibiting the expression of the HSD17B1 gene; (x3) a substance for overexpressing the NUCB2 gene; and, (x4 ) at least one of substances for inhibiting NUCB2 gene expression.
在一些可选的实施方式中，用于过表达HSD17B1基因的物质、用于抑制HSD17B1基因表达的物质、用于过表达NUCB2基因的物质和用于抑制NUCB2基因表达的物质分别独立的包括DNA、RNA、蛋白、多肽、化合物药物、细胞和微生物中的至少一种。一些可选的实例包括但不限于：用于干扰HSD17B1基因表达的siRNA，序列例如可以但不限于含有如SEQ IDNO.3、SEQ ID NO.4或SEQ ID NO.5所示序列，优选含有如SEQ ID NO.5所示序列；用于干扰NUCB2基因表达的siRNA，序列例如可以但不限于含有如SEQ ID NO.6、SEQ ID NO.7或SEQID NO.8所示序列，优选含有如SEQ ID NO.6所示序列；表达HSD17B1基因cDNA的载体，例如可以但不限于pIRES2-ZsGreen1-HSD17B1，以实现HSD17B1过表达；表达NUCB2基因cDNA的载体，例如可以但不限于pIRES2-ZsGreen1-NUCB2，以实现NUCB2过表达；能够表达干扰HSD17B1基因和/或NUCB2基因的siRNA的DNA，所述DNA可以是含有编码siRNA的表达盒或含有编码siRNA的载体，如重组质粒；能够抑制靶基因表达的蛋白、多肽或化合物药物，以实现对基因进行调控；能够表达具有调控基因作用的细胞或微生物，例如可以但不限于为含有能够表达siRNA或HSD17B1基因和/或NUCB2基因的重组载体的细胞或微生物。In some optional embodiments, the substance for overexpressing the HSD17B1 gene, the substance for inhibiting the expression of the HSD17B1 gene, the substance for overexpressing the NUCB2 gene and the substance for inhibiting the expression of the NUCB2 gene independently comprise DNA, At least one of RNA, protein, polypeptide, compound drug, cell and microorganism. Some optional examples include, but are not limited to: siRNA for interfering with HSD17B1 gene expression, the sequence may for example, but not be limited to, contain the sequence shown in SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. The sequence shown in SEQ ID NO.5; the siRNA for interfering with the expression of the NUCB2 gene, the sequence may, for example but not be limited to, contain the sequence shown in SEQ ID NO.6, SEQ ID NO.7 or SEQID NO.8, preferably the sequence shown in SEQ ID NO.8 The sequence shown in ID NO.6; the vector expressing HSD17B1 gene cDNA, such as but not limited to pIRES2-ZsGreen1-HSD17B1, to achieve HSD17B1 overexpression; the vector expressing NUCB2 gene cDNA, such as but not limited to pIRES2-ZsGreen1-NUCB2, To achieve NUCB2 overexpression; DNA capable of expressing siRNA that interferes with HSD17B1 gene and/or NUCB2 gene, the DNA can be an expression cassette containing siRNA encoding or a vector containing siRNA encoding siRNA, such as recombinant plasmids; proteins capable of inhibiting the expression of target genes , polypeptide or compound drugs to achieve gene regulation; cells or microorganisms capable of expressing the role of regulating genes, such as but not limited to cells or microorganisms containing recombinant vectors capable of expressing siRNA or HSD17B1 gene and/or NUCB2 gene.
本发明提供的颗粒细胞的调控方法，或用于调控颗粒细胞的产品可以用于调控卵泡发育或制备调控卵泡的产品中。颗粒细胞对卵泡和卵巢的发育具有调控作用：The method for regulating granulosa cells provided by the present invention, or the products for regulating granulosa cells can be used in regulating follicle development or preparing products for regulating follicles. Granulosa cells regulate the development of follicles and ovaries:
在卵泡发育过程中，颗粒细胞的增殖、分化和凋亡均会影响卵泡和卵巢的发育，有研究显示如果卵泡在发育时期颗粒细胞凋亡会导致卵泡闭锁，体外受精中颗粒细胞凋亡率低的胚胎移植后妊娠率更高。In the process of follicle development, the proliferation, differentiation and apoptosis of granulosa cells will affect the development of follicles and ovaries. Some studies have shown that if the follicles undergo apoptosis during the developmental period, granulosa cells will lead to follicular atresia, and the apoptosis rate of granulosa cells in in vitro fertilization is low. pregnancy rates after embryo transfer are higher.
颗粒细胞分泌的激素也参与调控卵泡和卵巢的发育：例如颗粒细胞分泌的INH对卵巢卵泡膜分泌雄激素具有促进作用；在卵泡发育的过程中INH促进受LH和IGF诱导的雄激素合成。颗粒细胞分泌的ACT具有促进卵泡成熟的作用，参与早期卵泡的发育，与成熟卵泡的闭锁有关。颗粒细胞分泌的FS与ACT结合后可阻止ACT的卵泡闭锁作用，促进卵泡继续发育成熟。颗粒细胞分泌的E2和P4也是具有多种作用的生殖相关激素，对卵巢发育具有多种作用。Hormones secreted by granulosa cells are also involved in the regulation of follicle and ovarian development: for example, INH secreted by granulosa cells promotes androgen secretion by ovarian theca; INH promotes androgen synthesis induced by LH and IGF during follicle development. ACT secreted by granulosa cells can promote follicle maturation, participate in the development of early follicles, and is related to the atresia of mature follicles. The combination of FS secreted by granulosa cells and ACT can prevent the follicular atresia effect of ACT and promote the continued development and maturation of follicles. E 2 and P 4 secreted by granulosa cells are also reproductive-related hormones with multiple effects on ovarian development.
从上述可以看出颗粒细胞对卵泡和/或卵巢的发育具有重要的调控作用，因此调控颗粒细胞可以进一步的用于调控卵泡和/或卵巢发育中，或用于制备调控卵泡和/或卵巢发育的产品中，例如可以为但不限于为调控卵泡和/或卵巢发育的试剂、试剂盒、药物或培养基等。It can be seen from the above that granulosa cells play an important role in regulating the development of follicles and/or ovaries. Therefore, regulating granulosa cells can be further used to regulate the development of follicles and/or ovaries, or to prepare and regulate the development of follicles and/or ovaries. Among the products, for example, it can be, but not limited to, reagents, kits, drugs or culture media for regulating follicle and/or ovarian development.
下面结合优选实施例进一步说明本发明的技术方案和有益效果。The technical solutions and beneficial effects of the present invention are further described below in conjunction with the preferred embodiments.
实施例1Example 1
(1)HSD17B1基因克隆与过表达：(1) HSD17B1 gene cloning and overexpression:
本实施例克隆了绵羊的HSD17B1基因全长957bp的cDNA序列，如SEQ ID NO.1所示。而后构建了羊源HSD17B1基因的真核表达载体：pIRES2-ZsGreen1-HSD17B1，并在绵羊卵巢颗粒细胞中瞬时转染验证其功能，其转染后HSD17B1基因的相对表达量为175.47±14.49％，如图1所示。In this example, the full-length 957bp cDNA sequence of the sheep HSD17B1 gene was cloned, as shown in SEQ ID NO.1. Then, the eukaryotic expression vector of sheep-derived HSD17B1 gene: pIRES2-ZsGreen1-HSD17B1 was constructed, and its function was verified by transient transfection in sheep ovarian granulosa cells. The relative expression of HSD17B1 gene after transfection was 175.47±14.49%, as shown in Figure 1.
(2)HSD17B1基因siRNA干扰：(2) HSD17B1 gene siRNA interference:
为了达到更好的siRNA干扰效果，首先针对绵羊的HSD17B1基因设计了3对干扰片段，siRNA-1、siRNA-2和siRNA-3依次分别含有SEQ ID NO.3～5所示序列，且3’端均分别独立的悬垂未配对的TT，如下所示：In order to achieve better siRNA interference effect, firstly, 3 pairs of interference fragments were designed for the HSD17B1 gene of sheep, siRNA-1, siRNA-2 and siRNA-3 respectively contain the sequences shown in SEQ ID NO. The ends are each independently overhanging the unpaired TT as follows:
HSD17B1 siRNA-1(3178)：5’-CCAGAGCUUCAAAGUGUAUtt-3’(SEQ ID NO.9)；HSD17B1 siRNA-1 (3178): 5'-CCAGAGCUUCAAAGUGUAUtt-3' (SEQ ID NO. 9);
HSD17B1 siRNA-2(3289)：5’-GGAUGUGAGGGAUGCAGAUtt-3’(SEQ ID NO.10)；HSD17B1 siRNA-2(3289): 5'-GGAUUGAGGGAUGCAGAUtt-3' (SEQ ID NO. 10);
HSD17B1 siRNA-3(3802)：5’-GGUGGUCGAGGUAAGUCUUtt-3’(SEQ ID NO.11)。HSD17B1 siRNA-3 (3802): 5'-GGUGGUCGAGGUAAGUCUUtt-3' (SEQ ID NO. 11).
结果如图1示，3对干扰片段干扰后HSD17B1基因相对空白组表达量分别为88.67±7.71％，77.07±4.03％和32.26±2.09％，因此，选择第3对干扰片段进行后续的细胞转染试验，转染后效果如图2所示。The results are shown in Figure 1. The expression levels of HSD17B1 gene relative to the blank group were 88.67±7.71%, 77.07±4.03% and 32.26±2.09% after the interference of the three pairs of interference fragments, respectively. Therefore, the third pair of interference fragments was selected for subsequent cell transfection Test, the effect after transfection is shown in Figure 2.
图1中“control”代表未经处理的对照组；“HSD17B1”代表HSD17B1基因经过表达处理或经干扰处理组。“OE”代表HSD17B1基因过表达处理组；“siRNA-1”代表HSD17B1基因siRNA-1处理组；“siRNA-2”代表HSD17B1基因siRNA-2处理组；“siRNA-3”代表HSD17B1基因siRNA-3处理组。数据表示为平均值±SEM，使用t-test进行统计分析。*代表差异显著，p＜0.05。In Figure 1, "control" represents the untreated control group; "HSD17B1" represents the HSD17B1 gene expression-treated or interference-treated group. "OE" stands for HSD17B1 gene overexpression treatment group; "siRNA-1" stands for HSD17B1 gene siRNA-1 treatment group; "siRNA-2" stands for HSD17B1 gene siRNA-2 treatment group; "siRNA-3" stands for HSD17B1 gene siRNA-3 processing group. Data are presented as mean ± SEM and statistical analysis was performed using t-test. *represents a significant difference, p<0.05.
图2中“siControl”代表siRNA空载对照组；“siHSD17B1”代表HSD17B1基因经干扰处理组。“ZsGreen1”代表绿色荧光镜下视野。标尺表示100μm。In Figure 2, "siControl" represents the siRNA empty control group; "siHSD17B1" represents the HSD17B1 gene interference treatment group. "ZsGreen1" represents the field of view under green fluorescence microscope. The scale bar indicates 100 μm.
实施例2Example 2
HSD17B1基因对绵羊卵巢颗粒细胞凋亡与增殖的影响：Effects of HSD17B1 gene on apoptosis and proliferation of sheep ovarian granulosa cells:
通过流式细胞凋亡检测，本实施例对HSD17B1基因过表达和干扰后的绵羊颗粒细胞凋亡情况进行了检测，发现HSD17B1基因干扰组颗粒细胞凋亡率5.50±0.21％，显著低于空白对照组(7.18±0.11％)，HSD17B1基因过表达组10.64±0.66％，明显高于空白对照组(7.18±0.11％)，如图3所示。此外，还通过MTT法对HSD17B1基因过表达和干扰后的绵羊颗粒细胞增殖率进行了检测，发现HSD17B1基因干扰组(102.89±3.99％)和过表达组(92.67±1.46％)都与空白组(100.00％)不存在显著差异，如图4和图5所示。Flow cytometry was used to detect the apoptosis of sheep granulosa cells after HSD17B1 gene overexpression and interference in this example. group (7.18±0.11%), the HSD17B1 gene overexpression group was 10.64±0.66%, which was significantly higher than that of the blank control group (7.18±0.11%), as shown in Figure 3. In addition, the proliferation rate of sheep granulosa cells after HSD17B1 gene overexpression and interference was also detected by MTT method, and it was found that the HSD17B1 gene interference group (102.89±3.99%) and the overexpression group (92.67±1.46%) were similar to the blank group ( 100.00%) were not significantly different, as shown in Figures 4 and 5.
图3～图5中“control”代表未经处理的对照组；“HSD17B1”代表HSD17B1基因经过表达处理或经干扰处理组。“siHSD17B1”代表HSD17B1基因siRNA干扰处理组；“OE”代表HSD17B1基因过表达处理组。数据表示为平均值±SEM，使用t-test进行统计分析。*代表差异显著,p＜0.05。In Figures 3 to 5, "control" represents the untreated control group; "HSD17B1" represents the HSD17B1 gene expression-treated or interference-treated group. "siHSD17B1" represents the HSD17B1 gene siRNA interference treatment group; "OE" represents the HSD17B1 gene overexpression treatment group. Data are presented as mean ± SEM and statistical analysis was performed using t-test. *represents a significant difference, p<0.05.
实施例3Example 3
HSD17B1基因对绵羊颗粒细胞E2、P4、ACT、INH和FS分泌的调节作用：Regulation of HSD17B1 gene on secretion of E 2 , P 4 , ACT, INH and FS in sheep granulosa cells:
为了验证HSD17B1基因对绵羊颗粒细胞E2和其他卵泡发育相关生殖激素的调节作用，通过ELISA方法对转染pIRES2-ZsGreen1-HSD17B1后颗粒细胞培养皿中的上清液进行了E2、P4、ACT、INH和FS检测，如图6～10所示。发现干扰组中，颗粒细胞对E2、P4和ACT的分泌量显著下降，INH和FS的分泌量显著升高；与此相对应的，过表达组中E2、P4和ACT的分泌量显著增加，而INH和FS的分泌量显著下降。说明HSD17B1基因能够在绵羊卵巢颗粒细胞中发挥功能，对细胞中的E2、P4和ACT的分泌具有促进作用，而对绵羊卵巢颗粒细胞中INH和FS的分泌具有抑制作用。In order to verify the regulatory effect of HSD17B1 gene on sheep granulosa cells E 2 and other reproductive hormones related to follicular development, the supernatants in granulosa cell culture dishes after transfection of pIRES2-ZsGreen1-HSD17B1 were subjected to E 2 , P 4 , ACT, INH and FS detection, as shown in Figures 6-10. It was found that in the interference group, the secretion of E 2 , P 4 and ACT by granulosa cells decreased significantly, and the secretion of INH and FS increased significantly; correspondingly, the secretion of E 2 , P 4 and ACT in the overexpression group The amount increased significantly, while the secretion of INH and FS decreased significantly. It indicated that HSD17B1 gene could function in ovary granulosa cells, promoting the secretion of E 2 , P 4 and ACT, and inhibiting the secretion of INH and FS in ovary granulosa cells.
图6～10中，“control”代表未经处理的对照组；“HSD17B1”代表HSD17B1基因经过表达处理或经干扰处理组。“siHSD17B1”代表HSD17B1基因siRNA干扰处理组；“OE”代表HSD17B1基因过表达处理组。数据表示为平均值±SEM，使用t-test进行统计分析。*代表差异显著，p＜0.05。In Figures 6-10, "control" represents the untreated control group; "HSD17B1" represents the HSD17B1 gene expression-treated or interference-treated group. "siHSD17B1" represents the HSD17B1 gene siRNA interference treatment group; "OE" represents the HSD17B1 gene overexpression treatment group. Data are presented as mean ± SEM and statistical analysis was performed using t-test. *represents a significant difference, p<0.05.
实施例4Example 4
(1)NUCB2基因克隆与过表达(1) NUCB2 gene cloning and overexpression
首先克隆了绵羊的NUCB2基因全长1,248bp的cDNA序列，序列如SEQ ID NO.2所示，而后构建了羊源NUCB2基因的真核表达载体：pIRES2-ZsGreen1-NUCB2，并在绵羊卵巢颗粒细胞中瞬时转染验证其功能，其转染后NUCB2基因的相对表达量为134.55±8.88％，如图11所示。First, the 1,248 bp cDNA sequence of the sheep NUCB2 gene was cloned, and the sequence is shown in SEQ ID NO. Its function was verified by transient transfection, and the relative expression of NUCB2 gene after transfection was 134.55±8.88%, as shown in Figure 11.
(2)NUCB2基因siRNA干扰(2) NUCB2 gene siRNA interference
为了达到更好的siRNA干扰效果，首先针对绵羊的NUCB2基因设计了3对干扰片段，siRNA-1、siRNA-2和siRNA-3依次分别含有SEQ ID NO.6～8所示序列，且3’端均分别独立的悬垂未配对的TT，如下所示：In order to achieve better siRNA interference effect, firstly, 3 pairs of interference fragments were designed for the NUCB2 gene of sheep. siRNA-1, siRNA-2 and siRNA-3 respectively contained the sequences shown in SEQ ID NO. The ends are each independently overhanging the unpaired TT as follows:
NUCB2 siRNA-1(368)：5’-GCUCUUGAAGCUGUGCCUAtt-3’(SEQ ID NO.12)；NUCB2 siRNA-1 (368): 5'-GCUCUUGAAGCUGUGCCUAtt-3' (SEQ ID NO. 12);
NUCB2 siRNA-2(739)：5’-CCUGAAUCCUAACAAGUUUtt-3’(SEQ ID NO.13)；NUCB2 siRNA-2(739): 5'-CCUGAAUCCUAACAAGUUUtt-3' (SEQ ID NO. 13);
NUCB2 siRNA-3(1295)：5’-CCAGAUAGCUGGGAGACAUtt-3’；(SEQ ID NO.14)。NUCB2 siRNA-3(1295): 5'-CCAGAUAGCUGGGAGACAUtt-3'; (SEQ ID NO. 14).
结果如图11所示，3对干扰片段干扰后NUCB2相对空白组表达量分别为53.14±5.63％，99.37±9.87％和71.37±12.30％，因此，选择第1对干扰片段进行后续的细胞转染试验。The results are shown in Figure 11. The expression levels of NUCB2 relative to the blank group after the interference of the three pairs of interference fragments were 53.14 ± 5.63%, 99.37 ± 9.87% and 71.37 ± 12.30%, respectively. Therefore, the first pair of interference fragments was selected for subsequent cell transfection test.
图11中“control”代表未经处理的对照组；“NUCB2”代表NUCB2基因经过表达处理或经干扰处理组。“OE”代表NUCB2基因过表达处理组；“siRNA-1”代表NUCB2基因siRNA-1处理组；“siRNA-2”代表NUCB2基因siRNA-2处理组；“siRNA-3”代表NUCB2基因siRNA-3处理组。数据表示为平均值±SEM，使用t-test进行统计分析。*代表差异显著,p＜0.05。In Figure 11, "control" represents the untreated control group; "NUCB2" represents the NUCB2 gene expression-treated or interference-treated group. "OE" stands for NUCB2 gene overexpression treatment group; "siRNA-1" stands for NUCB2 gene siRNA-1 treatment group; "siRNA-2" stands for NUCB2 gene siRNA-2 treatment group; "siRNA-3" stands for NUCB2 gene siRNA-3 processing group. Data are presented as mean ± SEM and statistical analysis was performed using t-test. *represents a significant difference, p<0.05.
实施例5Example 5
NUCB2基因对绵羊颗粒细胞凋亡与增殖的影响：Effects of NUCB2 gene on apoptosis and proliferation of sheep granulosa cells:
通过流式细胞凋亡检测，对NUCB2基因过表达和干扰后的绵羊颗粒细胞凋亡情况进行了检测，如图2所示。发现NUCB2基因干扰组颗粒细胞凋亡率4.36±0.15％显著低于空白对照组(6.98±0.07％)，过表达组12.78±0.30％显著高于空白对照组。还通过MTT法对NUCB2基因过表达和干扰后的绵羊颗粒细胞增殖率进行了检测，发现NUCB2基因干扰组细胞增殖率105.02±1.06％显著高于空白对照组(100.00％)，但过表达组(92.51±1.77％)与空白组(100.00％)不存在显著差异，如图13和图14所示。The apoptosis of sheep granulosa cells after NUCB2 gene overexpression and interference was detected by flow cytometry, as shown in Figure 2. It was found that the apoptosis rate of granulosa cells in the NUCB2 gene interference group was 4.36±0.15% significantly lower than that in the blank control group (6.98±0.07%), and the overexpression group was 12.78±0.30% significantly higher than the blank control group. The proliferation rate of sheep granulosa cells after NUCB2 gene overexpression and interference was also detected by MTT method, and it was found that the cell proliferation rate of NUCB2 gene interference group was 105.02±1.06%, which was significantly higher than that of blank control group (100.00%), but the overexpression group ( 92.51±1.77%) and the blank group (100.00%) were not significantly different, as shown in Figure 13 and Figure 14.
图12～图15中“control”代表未经处理的对照组；“NUCB2”代表NUCB2基因经过表达处理或经干扰处理组。“siNUCB2”代表NUCB2基因siRNA干扰处理组；“OE”代表NUCB2基因过表达处理组。数据表示为平均值±SEM，使用t-test进行统计分析。*代表差异显著，p＜0.05。In Figures 12 to 15, "control" represents the untreated control group; "NUCB2" represents the NUCB2 gene expression-treated or interference-treated group. "siNUCB2" represents the NUCB2 gene siRNA interference treatment group; "OE" represents the NUCB2 gene overexpression treatment group. Data are presented as mean ± SEM and statistical analysis was performed using t-test. *represents a significant difference, p<0.05.
实施例6Example 6
NUCB2基因对绵羊颗粒细胞p-AKT蛋白表达的影响：The effect of NUCB2 gene on the expression of p-AKT protein in sheep granulosa cells:
由于NUCB2基因参与编码Nestfin-1蛋白，Nestfin-1蛋白可通过AKT信号通路起作用，所以对NUCB2基因过表达和干扰后的绵羊颗粒细胞AKT信号通路标志性蛋白p-AKT表达量进行了Western Blot检验。Western Blot实验结果表明NUCB2基因过表达，能显著降低细胞中p-AKT的水平，如图15和图16所示。Since the NUCB2 gene is involved in encoding the Nestfin-1 protein, and the Nestfin-1 protein can act through the AKT signaling pathway, Western Blot was performed on the expression of p-AKT, a marker protein of the AKT signaling pathway in sheep granulosa cells after the overexpression and interference of the NUCB2 gene. test. The results of Western Blot experiments showed that overexpression of NUCB2 gene could significantly reduce the level of p-AKT in cells, as shown in Figure 15 and Figure 16 .
图15使用BandScan 5.0软件进行灰度扫描分析，“siNUCB2”代表NUCB2基因干扰处理组；“OE”代表NUCB2基因过表达处理组；“control”代表未经处理的对照组；“NUCB2”代表NUCB2基因经过表达处理或经干扰处理组；数据表示为平均值±SEM，使用t-test进行统计分析。*代表差异显著，p＜0.05。Figure 15 Grayscale scanning analysis using BandScan 5.0 software, "siNUCB2" represents the NUCB2 gene interference treatment group; "OE" represents the NUCB2 gene overexpression treatment group; "control" represents the untreated control group; "NUCB2" represents the NUCB2 gene Expression-treated or perturbed-treated groups; data are presented as mean ± SEM, and statistical analysis was performed using t-test. *represents a significant difference, p<0.05.
图16中“p-AKT”代表NUCB2基因干扰或过表达处理组；GAPDH为内参；“si-NUCB2”代表NUCB2基因干扰处理组；“OE”代表NUCB2基因过表达处理组；“control”代表未经处理的对照组；“si-NC”代表NUCB2基因干扰空载组；“si-NUCB2”代表NUCB2基因干扰处理组；“over-NC”代表NUCB2基因过表达空载组。In Figure 16, "p-AKT" represents the NUCB2 gene interference or overexpression treatment group; GAPDH is the internal control; "si-NUCB2" represents the NUCB2 gene interference treatment group; "OE" represents the NUCB2 gene overexpression treatment group; The treated control group; "si-NC" represents the NUCB2 gene interference null group; "si-NUCB2" represents the NUCB2 gene interference treatment group; "over-NC" represents the NUCB2 gene overexpression null group.
实施例7Example 7
NUCB2基因对绵羊颗粒细胞E2、P4、ACT、INH和FS分泌的调节作用：Regulation of NUCB2 gene on E 2 , P 4 , ACT, INH and FS secretion in sheep granulosa cells:
为了验证NUCB2基因对绵羊颗粒细胞E2和其他卵泡发育相关生殖激素的调节作用，通过ELISA方法对转染pIRES2-ZsGreen1-NUCB2后颗粒细胞培养皿中的上清液进行了E2、P4、ACT、INH和FS，结果如图17～21所示。发现干扰组中，颗粒细胞对E2、P4和ACT的分泌量显著增高，INH和FS的分泌量显著下降；与此相对应的，过表达组中E2、P4和ACT的分泌量显著降低，而INH和FS的分泌量显著上升。说明NUCB2基因能够在绵羊卵巢颗粒细胞中发挥功能，对细胞中的E2、P4和ACT的分泌具有抑制作用，而对绵羊颗粒细胞中INH和FS的分泌具有促进作用。In order to verify the regulatory effect of NUCB2 gene on sheep granulosa cells E2 and other reproductive hormones related to follicle development, E 2 , P 4 , ACT were carried out on the supernatant of granulosa cell culture dishes after transfection of pIRES2-ZsGreen1-NUCB2 by ELISA method. , INH and FS, the results are shown in Figures 17-21. It was found that in the interference group, the secretion of E 2 , P 4 and ACT by granulosa cells was significantly increased, and the secretion of INH and FS was significantly decreased. Correspondingly, the secretion of E 2 , P 4 and ACT in the overexpression group significantly decreased, while the secretion of INH and FS increased significantly. It indicated that NUCB2 gene could function in ovary granulosa cells, inhibiting the secretion of E 2 , P 4 and ACT in the cells, and promoting the secretion of INH and FS in ovine granulosa cells.
图17～21中，“control”代表未经处理的对照组；“NUCB2”代表NUCB2基因干扰或过表达处理组；“siNUCB2”代表NUCB2基因干扰处理组；“OE”代表NUCB2基因过表达处理组。数据表示为平均值±SEM，使用t-test进行统计分析。*代表差异显著，p＜0.05。In Figures 17-21, "control" represents the untreated control group; "NUCB2" represents the NUCB2 gene interference or overexpression treatment group; "siNUCB2" represents the NUCB2 gene interference treatment group; "OE" represents the NUCB2 gene overexpression treatment group . Data are presented as mean ± SEM and statistical analysis was performed using t-test. *represents a significant difference, p<0.05.
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features thereof can be equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention. scope.
SEQUENCE LISTINGSEQUENCE LISTING
<110> 吉林省农业科学院<110> Jilin Academy of Agricultural Sciences
<120> HSD17B1基因和/或NUCB2基因的应用和颗粒细胞的调控方法<120> Application of HSD17B1 gene and/or NUCB2 gene and regulation method of granulosa cells
<160> 14<160> 14
<170> PatentIn version 3.5<170> PatentIn version 3.5
<210> 1<210> 1
<211> 954<211> 954
<212> DNA<212> DNA
<213> 绵羊（Ovis aries）<213> Sheep (Ovis aries)
<400> 1<400> 1
atggaccgga ccgtggtgct gatcaccggg tgcagcagcg ggatcgggct gcacctggcc 60atggaccgga ccgtggtgct gatcaccggg tgcagcagcg ggatcgggct gcacctggcc 60
ctgaggctgg cctctgaccc cagccagagc ttcaaggtgt acgccaccct gagggacctg 120ctgaggctgg cctctgaccc cagccagagc ttcaaggtgt acgccaccct gagggacctg 120
gcctctcagg gacctctcct ggaagctgcc cagagcaggg gctgcccccc tggaagtctg 180gcctctcagg gacctctcct ggaagctgcc cagagcaggg gctgcccccc tggaagtctg 180
gagaccctgc agctggacgt gagggacgcc gacagtatcg ccgccgccca ggctagggtg 240gagaccctgc agctggacgt gagggacgcc gacagtatcg ccgccgccca ggctagggtg 240
accgagggaa gagtggacgt gctggtgtgc aacgcaggaa ggggcctggt gggacctctg 300accgagggaa gagtggacgt gctggtgtgc aacgcaggaa ggggcctggt gggacctctg 300
gaggctcata aggagggcag cgtggacgcc gtgctggacg tgaacctgac cggaaccgtg 360gaggctcata aggagggcag cgtggacgcc gtgctggacg tgaacctgac cggaaccgtg 360
aggatgctgc aggcatttct gcctgacatg aagaggagga gaagcggaag gatcctggtg 420aggatgctgc aggcatttct gcctgacatg aagaggagga gaagcggaag gatcctggtg 420
accggcagca tcggaggact gatggccctg cccttcaacg ccgtgtactg tgccagcaag 480accggcagca tcggaggact gatggccctg cccttcaacg ccgtgtactg tgccagcaag 480
ttcgccctcg aaggcctctg cgagagcctg gcaatcctgc tgcagccatt cggcgtgcat 540ttcgccctcg aaggcctctg cgagagcctg gcaatcctgc tgcagccatt cggcgtgcat 540
gtgtccctga tcgaatgcgg ccccgtgcat acccccttcc ccgagaaggt ggaaggcggt 600gtgtccctga tcgaatgcgg ccccgtgcat acccccttcc ccgagaaggt ggaaggcggt 600
ctgggcggaa tgctcgacag ggccgacgcc gagacccgcg acctgtttta taggtactgt 660ctgggcggaa tgctcgacag ggccgacgcc gagacccgcg acctgtttta taggtactgt 660
aggcattgcg agagggtgat cagggaggca ggacaggacc cagaggaggt ggtggaggtg 720aggcattgcg agagggtgat cagggaggca ggacaggacc cagaggaggt ggtggaggtg 720
ttcctgcagg ccctgagggc ccccagacca gcactgaggt acttcaccac cgagcggttc 780ttcctgcagg ccctgagggc ccccagacca gcactgaggt acttcaccac cgagcggttc 780
ctccccctgg tgcagctgag atttagcgac cccagtggca gcgactatgt ggcagccgcc 840ctccccctgg tgcagctgag atttagcgac cccagtggca gcgactatgt ggcagccgcc 840
cataaaagcg ccttccacga caaggccgcc gaaggatccg acggcaacgg agctgaggcc 900cataaaagcg ccttccacga caaggccgcc gaaggatccg acggcaacgg agctgaggcc 900
gaagccggca aactgggcgc cagcgaactg agggctcccc tggctcctca gtag 954gaagccggca aactgggcgc cagcgaactg agggctcccc tggctcctca gtag 954
<210> 2<210> 2
<211> 1248<211> 1248
<212> DNA<212> DNA
<213> 绵羊（Ovis aries）<213> Sheep (Ovis aries)
<400> 2<400> 2
atgaagtgga ggaccatcct gccccagtgc tgcttcttcc tggtgacctg cgccctgacc 60atgaagtgga ggaccatcct gccccagtgc tgcttcttcc tggtgacctg cgccctgacc 60
gccctggagg ctgtgcctat cgacatcgac aagaccaagg tgaaggccgc ccagcccgtg 120gccctggagg ctgtgcctat cgacatcgac aagaccaagg tgaaggccgc ccagcccgtg 120
gacagcgcta aaatcgaacc ccccgacacc ggcctctact acgacgagta tctcaagcag 180gacagcgcta aaatcgaacc ccccgacacc ggcctctact acgacgagta tctcaagcag 180
gtgatcgacg tgctcgagac cgacagccac ttcagagaga aactgcagaa ggccgacatc 240gtgatcgacg tgctcgagac cgacagccac ttcagagaga aactgcagaa ggccgacatc 240
gaggagatca agagcggccg cctgagcaag gaactggacc tggtgagcca tcatgtgaga 300gaggagatca agagcggccg cctgagcaag gaactggacc tggtgagcca tcatgtgaga 300
accaaactgg acgagctgaa aaggcaggag gtggcaaggc tgaggatgct gatcaaggca 360accaaactgg acgagctgaa aaggcaggag gtggcaaggc tgaggatgct gatcaaggca 360
aagctggaca gcttccagga caccggcatc gaccatcatg ccctgctcaa acagttcgac 420aagctggaca gcttccagga caccggcatc gaccatcatg ccctgctcaa acagttcgac 420
cacctgaacc atctgaaccc caacaagttc gaatccaccg acctggacat gctgatcaaa 480cacctgaacc atctgaaccc caacaagttc gaatccaccg acctggacat gctgatcaaa 480
gccgccacct ccgacctgga acattacgac aaggccagac atgaagagtt taagaagtac 540gccgccacct ccgacctgga acattacgac aaggccagac atgaagagtt taagaagtac 540
gaaatgatga aagaacacga gagaagggag tacctgaaaa ccctgagcga agagaagaga 600gaaatgatga aagaacacga gagaagggag tacctgaaaa ccctgagcga agagaagaga 600
aaggaagagg agagtaagtt cgccgagatg aagaagaagc atgagaacca tccaaaggtg 660aaggaagagg agagtaagtt cgccgagatg aagaagaagc atgagaacca tccaaaggtg 660
aaccatccag gaagcaagga ccagctgaag gaagtgtggg aggaggcaga cggcctggac 720aaccatccag gaagcaagga ccagctgaag gaagtgtggg aggaggcaga cggcctggac 720
ccaaacgact tcgaccccaa gaccttcttc aagctgcatg acgtgaacag cgacggcttc 780ccaaacgact tcgaccccaa gaccttcttc aagctgcatg acgtgaacag cgacggcttc 780
ctcgacgagc aggagctgga ggccctgttt accaaggagc tggagaaggt gtacgacccc 840ctcgacgagc aggagctgga ggccctgttt accaaggagc tggagaaggt gtacgacccc 840
aaaaacgagg aagacgacat ggtggagatg gaagaggaga ggctcaggat gagggagcat 900aaaaacgagg aagacgacat ggtggagatg gaagaggaga ggctcaggat gagggagcat 900
gtgatgaacg aagtggacac caacaaggac aggctggtga ccctggagga gttcctgaag 960gtgatgaacg aagtggacac caacaaggac aggctggtga ccctggagga gttcctgaag 960
gcaaccgaaa agaaggagtt cctggagcca gacagctggg agaccctgga ccagcagcag 1020gcaaccgaaa agaaggagtt cctggagcca gacagctggg agaccctgga ccagcagcag 1020
ttcttcaccg aggaggagct gaaagagtac gagaacctga tcagcgtgca ggagagcaag 1080ttcttcaccg aggaggagct gaaagagtac gagaacctga tcagcgtgca ggagagcaag 1080
ctcaaggaaa aggcagacga gctgcagaag cagaaggagg acctgcagag gcagcacgag 1140ctcaaggaaa aggcagacga gctgcagaag cagaaggagg acctgcagag gcagcacgag 1140
cagctggagg cacagaagct ggagtaccat caggtggtgc agcagatgga gcagaagaag 1200cagctggagg cacagaagct ggagtaccat caggtggtgc agcagatgga gcagaagaag 1200
ctgcagcaga tcagccccag cgcccccgga ggagagagca aaatctag 1248ctgcagcaga tcagccccag cgccccccgga ggagagagca aaatctag 1248
<210> 3<210> 3
<211> 19<211> 19
<212> RNA<212> RNA
<213> 人工序列<213> Artificial sequences
<400> 3<400> 3
ccagagcuuc aaaguguau 19ccagagcuuc aaaguguau 19
<210> 4<210> 4
<211> 19<211> 19
<212> RNA<212> RNA
<213> 人工序列<213> Artificial sequences
<400> 4<400> 4
ggaugugagg gaugcagau 19ggaugugagg gaugcagau 19
<210> 5<210> 5
<211> 19<211> 19
<212> RNA<212> RNA
<213> 人工序列<213> Artificial sequences
<400> 5<400> 5
gguggucgag guaagucuu 19gguggucgag guaagucuu 19
<210> 6<210> 6
<211> 19<211> 19
<212> RNA<212> RNA
<213> 人工序列<213> Artificial sequences
<400> 6<400> 6
gcucuugaag cugugccua 19gcucuugaag cugugccua 19
<210> 7<210> 7
<211> 19<211> 19
<212> RNA<212> RNA
<213> 人工序列<213> Artificial sequences
<400> 7<400> 7
ccugaauccu aacaaguuu 19ccugaauccu aacaaguuu 19
<210> 8<210> 8
<211> 19<211> 19
<212> RNA<212> RNA
<213> 人工序列<213> Artificial sequences
<400> 8<400> 8
ccagauagcu gggagacau 19ccagauagcu gggagacau 19
<210> 9<210> 9
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<400> 9<400> 9
ccagagcuuc aaaguguaut t 21ccagagcuuc aaaguguaut t 21
<210> 10<210> 10
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<400> 10<400> 10
ggaugugagg gaugcagaut t 21ggaugugagg gaugcagaut t 21
<210> 11<210> 11
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<400> 11<400> 11
gguggucgag guaagucuut t 21gguggucgag guaagucuut t 21
<210> 12<210> 12
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<400> 12<400> 12
gcucuugaag cugugccuat t 21gcucuugaag cugugccuat t 21
<210> 13<210> 13
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<400> 13<400> 13
ccugaauccu aacaaguuut t 21ccugaauccu aacaaguuut t 21
<210> 14<210> 14
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<400> 14<400> 14
ccagauagcu gggagacaut t 21ccagauagcu gggagacaut t 21
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