CN107406844A - The transgenic rice plant of improvement - Google Patents

Abstract

The present invention relates to rice specificity Microrna, i.e. miR820, by the gene that tiller and Floral development are effectively controlled from the Gene Isolation of India's Oryza species, clone and functional verification.It is connected the present invention relates to nucleotide sequence (nucleotide fragments containing miR820) with exogenous promoter, and wherein promoter is alternatively constitutive promoter or inducible promoter, and it is introduced into long-grained nonglutinous rice cultivar.Compared with the wild-type plant cultivated under the same conditions, transgenic rice plant has the agronomic characteristics of improvement, including bigger tiller, abundant fringe and more flowers.

Description

Improved transgenic rice plants

technical field

The invention relates to the fields of plant genetics and plant biotechnology. In particular, the present invention provides improved transgenic plants comprising plant cells having recombinant DNA encoding regulatory RNAs that negatively regulate endogenous proteins to result in the expression of improved agronomic traits. In particular, the present invention relates to the overexpression of rice-specific amiRNA (artificial microRNA) using genetic engineering techniques, which leads to rice plants with increased height, tolerance to salt stress, and increased grain yield.

Background technique

Plant transformation is an important tool for understanding various aspects of genes and their roles in plants. For commercial purposes, this information can be used to generate improved crop varieties. Rice is an important cereal crop that feeds half of the world's population. However, rice is a sensitive crop and its yield is affected by abiotic stresses, such as salinity, high temperature, drought stress, pathogens and pests. Several methods of transformation in rice have been reported. However, many of these methods are unpredictable in terms of transformation efficiency and regenerative capacity of explants.

MicroRNAs (miRNAs) are small endogenous RNAs that regulate gene expression in plants and animals. miRNA gene transcription produces primary miRNA (pri-miRNA) transcripts, which are processed into shorter hairpin loop structures of 20-60 nucleotides in length, called precursor miRNAs (pre-miRNAs). The precursor miRNA (pre-miRNA) is further processed to generate a duplex containing the mature miR. In plants, they are processed by Dicer-like enzymes from the stem-loop region of long precursor miRNA (pre-miRNA) transcripts and loaded into the silencing complex, where they are usually directly cut (off, switching off) complementary mRNA.

Thus, microRNAs (miRNAs) are a newly identified class of endogenous non-coding RNA molecules that play a major role in genome dynamics at the molecular level. They can regulate target gene expression in a sequence-dependent manner. MicroRNAs (miRNAs) regulate gene expression through three distinct mechanisms: (a) cleavage of complementary target mRNAs, (b) repression of translation of target mRNAs, and (c) transcriptional silencing of target mRNAs. MicroRNAs (miRNAs) are known to play many critical roles at every major stage of development, often at the heart of gene regulatory networks, targeting genes that are themselves regulators. Although plant miRNAs have some conserved functions that extend beyond development, the importance of miRNA-guided gene regulation during plant development is now becoming clear. So far, microRNAs have been found to be involved in plant development, regulation of abiotic and biotic stress responses, and hormone signaling (Jones-Rhoades et al., 2006, Ann Rev Plant Biol 57: 19-53).

Plants display remarkable plasticity by regulating development in response to changes in various environmental conditions. This requires rapid transient or stable transcriptional reprogramming to regulate plant responses. Plant microRNAs (miRNAs) have become important regulators of gene expression in normal and stressful environments. Rice is a major cereal crop and its production is greatly threatened under the current global climate change situation. That is, productivity is compromised due to various abiotic stress factors such as salt, heat and drought. Rice transformation is a challenging task because it is relatively not amenable to genetic manipulation. Therefore, it is important to generate cultivars that can sustain yields in limited arable land under deteriorating climatic conditions. MicroRNAs are now known to be one of the major factors contributing to genome regulation for plant growth and development and for coping with stressful environments. Cataloging microRNAs in rice is an important step in understanding the physiology of this important cereal crop under metabolic or environmental stress.

Accordingly, there is a need to develop transgenic plants with various improved agronomic traits, particularly plants that are viable and produce high yield and express other desirable traits in Oryza species. This will go a long way towards meeting the challenge of changing climatic conditions to increase demand for food products such as Oryza in rice-consuming countries.

purpose of invention

The object of the present invention is to provide improved transgenic rice plants with improved agronomic traits such as high yield and tolerance to salt stress. The present invention also provides a method of improving plant vigor and other agronomic characteristics of plants in Oryza species.

Description of drawings

Further illustrate the present invention below with reference to accompanying drawing:

Figure 1 is the pCAMBIA1300-amiR820 construct containing the amiR820 cassette.

Figure 2 compares transgenic plants overexpressing Osa-miR820 (OX-820) compared to untransformed wild-type (WT) rice plants. Overexpressing plants exhibited longer plant height with more tillers compared to non-transformed wild-type (WT) rice plants. The left panel (Panel) 1 shows the morphology of whole OX-820 rice plants and wild-type (WT) rice plants. Plant A was overexpressing OX-820, which showed increased plant height of the plants. Plant B shows the plant height of wild type plant Pusa basmati 1 (PB1 ), which has a shorter height compared to Ox-820. Right experimental group 2 shows more grain-filled (drooping) ears in OX-820 compared to wild-type (WT) rice plants. Plant C shows more drooping ears in OX-820 and plant D shows wild type plants Pusabasmati1 (PB1 ) with less drooping panicles compared to OX-820.

Figure 3 compares transgenic plants (OX-820) overexpressing Osa-miR820 with untransformed plants. Transgenic plants showed increased ear length, more abundant branches of ears, and enhanced grain filling on individual ear branches. Experimental group 1 shows plant A overexpressing OX-820 with increased ear length, and plant B is WTPusa basmati 1 (PB1 ) with shorter ear length compared to OX-820. Experimental group 2 showed plants C OX-820 with more ear branches, and wild type (WT) plant D showed less ear branches. Experimental group 3 showed plant E OX-820 with more grains, and plant F wild type (WT) showed less number of grains compared to OX-820.

detailed description

The present invention relates to transgenic rice plants employing recombinant deoxyribonucleic acid (DNA) for the expression of RNA useful for conferring improved agronomic traits and tolerance to salt stress. In particular, the present invention provides transgenic rice plants in which osa-miR820 is overexpressed, whereby transgenic rice cultivars (cultivar, varieties) are not only moderately tolerant to salt stress, but also have higher plant height and higher grain yield. very high.

The present invention also provides methods for developing transgenic seeds that can be used to generate transgenic rice plants with improved plant traits resulting from overexpression of osa-miR820.

In one aspect, a construct comprising the mature sequence of osa-miR820 is developed and cloned into a suitable vector. Plant cells were transformed using Agrobacterium-mediated transformation and microprojectile bombardment. For Agrobacterium-based plant transformation, additional elements may be present on the transformation construct, including T-DNA left and right border sequences, to facilitate incorporation of the recombinant polynucleotide into the plant genome. Typically, recombinant DNA can be introduced randomly, ie at non-specific locations in the genome of the target plant line. Analysis by PCR using appropriate primers such as 820-Fwd with SEQ ID No: 7-: 5'-TCGGCCTCGTGGATGG-3' and 820-Rev with SEQ ID No. 8-: 5'-GTGCAGGGTCCGAGGT-3' to confirm the conversion.

In another aspect, transformed cells are grown using tissue culture techniques in a controlled environment using a suitable plant growth nutrient medium that promotes cell growth. Calli from this tissue culture are grown into mature fertile transgenic plants and seeds are harvested. These seeds can be used to grow progeny of transformed plants of the invention, including hybrid plant lines for selection of plants expressing improved agronomic traits. Transgenic plants with recombinant DNA and expressing improved agronomic traits have increased plant height, more tillers, grain yield and tolerance to salt stress. The resulting transgenic plants are identified by selection for plants of particular interest expressing improved agronomic traits.

In one aspect, the present invention relates to the important role played by Osa-miR820 (rice-specific microRNA). Osa-miR820 processed by Dicer-like protein (DCL1) or Dicer-like protein (DCL3) generated sequences of 21 nucleotides and 24 nucleotides. The present inventors have discovered that miR820 has a dual function, in which the 21 nucleotide species plays a role in reverse guide agronaute (AGO1)-mediated cleavage of DNA (deoxyribonucleic acid) methyltransferase, while the 24 nucleotide Acid species act through an agrannute (AGO4)-mediated pathway to establish domain rearrangement methyltransferase (OsDRM2) and its own epigenetic modification of the site. One of the inventors' findings was that overexpression of miR820 produced variants of 21 nucleotides and 24 nucleotides and thus masked the effect of PTGS (post-transcriptional gene regulation).

In one aspect, the full benefit and effect of overexpression of miR820 was achieved with some changes in the medium used for callus regeneration. The usual vegetative growth medium is modified using a combination of vitamins, sucrose, and antibiotics.

In one embodiment, the following medium supports the growth of callus regeneration (see Table 1)

Table 1: Effect of different media on regeneration of callus transformed with pCAMBIA1300-amiR820 construct

The modified medium was able to grow regenerants in less than 7 days.

The transgenic plants of the invention exhibit increased yield compared to control plants. An increase in yield was found to be greater than 7-10% compared to control plants and was also attributed to the presence of osa-miR820. Increased yield was determined by the increase in ear number seen in transformed plants. The increase in ears is at least 10% and the grain yield is increased by 7-10% per hectare compared to control plants.

Efficient yield screening is accomplished by using hybrid progeny of transgenic events at multiple locations where plants are grown under optimal production techniques, and using positive and negative control plants at multiple locations and two growing seasons .

On the other hand, it was found that the transgenic plants of the present invention not only exhibit increased yield, but are also tolerant to salt stress. Tolerance to salt stress was determined by short- and long-term stress treatments. Short-term stress treatments were measured using a chlorophyll retention assay as a marker of delayed stress-induced senescence. This is a rapid assay that assesses the amount of chlorophyll retained in isolated leaves as a marker of cell death. The occurrence of natural senescence or stress-induced senescence is known to be associated with loss of chlorophyll content, so this parameter has been used to assess the performance of transgenics under salt and other abiotic stresses. To understand the role of OX-820 under salinity stress, germination assays of OX-820 rice seeds were performed at different NaCl concentrations, while wild-type (WT) seeds were used as controls. It was observed that OX-820 germination was comparable to wild type (WT) at all concentrations of sodium chloride (NaCl).

The growth performance of OX-820 seedlings under salt stress was analyzed by measuring the total fresh weight, shoot length and root length of 1-week-old seedlings grown under normal (water) and salt stress conditions. It was observed that OX-820 seedlings performed much better than wild-type (WT) seedlings under normal stress-free conditions. However, in the presence of 100 mM and 200 mM sodium chloride (NaCl), OX-820 seedlings performed similarly to WT seedlings relative to unstressed plants, both showing stress-induced decreases in all parameters . Therefore, it is clear that OX-820 seedlings can tolerate mild salinity conditions like their wild-type (WT) counterparts, but not higher salt concentrations.

Long-term assays involve growing plants to maturity in the presence of high soil salts. It was observed that under control conditions, OX-820 plants were taller and had higher tiller numbers compared to wild type (WT), but under salt stress conditions, OX-820 plants showed a significant decrease in plant height, Plant height is now comparable to that of unstressed wild-type (WT) plants. There was not much drop in ear number and less compensation for ear characteristics. On the other hand, wild type (WT) showed no reduction in plant height, but a dramatic reduction in the number of primary tillers. Therefore, it is clear that OX-820 plants showed better vigor under control as well as under salt stress conditions.

In yet another aspect, the transgenic plants of the invention also have an increased level of branching in the plant, which serves as a major determinant of improved plant height, root biomass and ear vigor. Tillering in rice is an important agronomic trait and it is positively correlated with grain yield in rice. Observations of improved tillering ability in control and salt-stressed OX-820 plants were then assessed against various ear-related traits. Higher grain yield related parameters such as ear length, total number of ears/plant and total ear weight/plant were found to be negatively affected by salt stress in OX-820 plants grown under control conditions. However, the degree of reduction was much smaller than that obtained in salt-stressed wild-type (WT) plants. Thus, phenotypic observations indicated that overexpression of Osa-miR820 increased overall rice productivity and decreased salt-stress-induced yield even though it moderately increased plant salt-stress responsiveness.

The expression of Osa-miR820 in rice cultivars can also be determined by analyzing leaf and ear tissues. This analysis revealed higher concentrations of the 21 nucleotide species Osa-miR820 in halotolerant plants. Furthermore, careful inspection of the expression pattern of the Osa-miR820-length variant revealed that the 21-nucleotide microRNA was downregulated in transgenic plants, resulting in a relative up-regulation of the 24-nucleotide-length variant. The inventors hypothesize that although the expression of Osa-miR820 is deregulated by salt stress, the ratio between the 21-nucleotide-long variant and the 24-nucleotide-long variant plays an important role and is responsible for the development of Tolerant to salt stress.

Furthermore, the expression levels of miR820 at the late flowering stage were ascertained and high quality spikelets were found to have a large amount of miR820 compared to poor quality spikelets, and this overexpression may be responsible for extensive panicle branching.

Analysis of 14-day-old transgenic seedlings also revealed very high levels of Osa-miR820 compared to untransformed wild seedlings.

The present invention relates to a method for increasing plant vigor by overexpressing miR820 by plant miRNA technology using selective nucleotide fragments and expression vectors. The overexpression method of rice-specific miR820 of the present invention comprises the following steps-

i) selection and growth of plants under optimal conditions;

ii) designing the plasmid construct;

iii) Cloning and expression of Osa-miR820;

iv) Agroinfiltration;

v) isolating RNA;

vi) Confirmation of the expressed transformed strain;

vii) Callus induction.

The steps are listed here as follows:

i) Selection and growth of plants under optimal conditions: Mature husked rice seeds can be surface sterilized with ethanol. The seeds can then be washed thoroughly with sterile distilled water and blotted dry.

ii) Design of plasmid constructs: Constructs can be formed from the backbone of Osa-miR528, a precursor of miRNAs conserved in Oryza species. The mature sequence of miR528 may be replaced by the mature sequence of Osa-miR820. The entire sequence can be cloned into a vector from the group comprising pGEMT-EASY, pUC19, pBR322, pRT101, preferably pRT101 can be used. Different combinations of primers can be used to generate PCR fragments. These fragments can be gel purified to obtain amplification products.

iii) Cloning and expression of Osa-miR820: Plant cells can be transformed from the group including Agrobacterium-mediated gene transfer, Agro-infection mediated, direct gene transfer, preferably using Agrobacterium-mediated Bacteria-mediated gene transfer. The entire cassette can be incorporated into an expression vector from the group comprising pSOMI, pet28a, pTAG, FLAG, pCAMBIA1300, preferably pCAMBIA1300 can be used.

Cloning of Osa-miR820 into the Agrobacterium tumefaciens binary vector pCAMBIA1300 can be achieved by cloning the amplified PCR product into pRT101 using EcoRI and BamHI under the CaMV-35S promoter. The entire cassette was excised from pRT101-Osa-miR820 by restriction digestion with HindIII and transformed into the pCAMBIA1300 binary vector using restriction enzymes. Recombinant pCAMBIA1300-amiR820 was confirmed by PCR and restriction digest. The plasmids were transformed into Agrobacterium tumefaciens strains EHA105 and LBA4404, and positive colonies were screened and selected for rice transformation. To confirm transformation, PCR was performed using specific primers and restriction digest.

iv) Agroinfiltration: Agroinfiltration can be achieved by pressure infiltration. Agrobacterium cultures can be grown overnight in nutrient media with appropriate antibiotics. Cells can be pelleted by brief centrifugation and resuspended in appropriate medium. Cells can be incubated (cultured) and then diluted in buffer. The homogenous culture mixture can be infiltrated into young leaves with the help of a needle-free syringe by creating a vacuum with the help of fingers on the backside of the leaf and the mouth of the syringe on the ventral side. After a few days, the infiltrated areas can be analyzed for miR820 expression.

v) Isolation of RNA: Transformed positive rice lines were used by various techniques from the group including organic extraction method, filter-based and spin basket method, magnetic particle method, direct lysis method Total RNA is extracted, preferably by organic extraction methods such as guanidine isothiocyanate. Plant tissue can be homogenized in liquid nitrogen, and GITC buffer can be added with phenol and chloroform. The mixture can be centrifuged at high speed. The aqueous phase can be obtained using an extraction solution, followed by precipitation. RNA pellets can be washed with DEPC-ethanol and stored for longer use. The presence of miR820 can be confirmed by sequencing.

vi) Confirmation of overexpressed transformed lines: Genomic DNA can be extracted from transgenic rice lines and wild rice lines. Plant material may be crushed into liquid nitrogen and homogenized. Homogenized samples can be saved for incubation. The mixture can be centrifuged and the supernatant collected in a new tube. The final pellet can be dried at room temperature and dissolved in buffer. PCR analysis can be performed and the amplified products can be examined. Plant material can be crushed and homogenized. The mixture can be centrifuged and the supernatant collected. The final pellet can be washed with alcohol, dried and dissolved in buffer. PCR can be performed followed by blotting to confirm expression of positive rice lines. Genomic DNA from PCR-positive rice lines digested with KpnI can be blotted on HybondN membranes. The hygromycin gene can be used as a probe, and hybridization can be performed.

vii) Callus induction: Surface sterilized seeds of rice cultivar Pusa basmati 1 (PB1 ) can be dried on autoclaved Whatman paper and grown in the dark on callus induction medium ( CIM) on the incubation. Calli can be excised and subcultured on fresh CIM in the dark. For co-infection, subcultured calli can be submerged in the Agrobacterium suspension with continuous slow shaking. After infection, calli can be blotted dry on sterile filter paper and can be incubated in the dark on filter paper moistened with co-cultivation medium (CCM). After co-cultivation, calli were washed and dried on sterile filter paper, and cultured on callus selection medium (CSM) in the dark to select for transgenic calli. After 20 days of the first round of selection, white calli can be transferred to fresh callus selection medium (CSM) medium for a second selection cycle of 15 days. Healthy callus can be selected for regeneration. After the third selection, healthy calli were transferred to specific regeneration media (RM1-4) and incubated in the dark in a culture room for 7 days. Green shoots can be seen emerging from the callus. Green shoots can develop into shoots and can be transferred to rooting medium (RoM) in the presence of hygromycin for 20 days in the light. Whole plants can be transferred to vermiculite pots, then to soil pots, and can be grown in a greenhouse. Certain modified CIM, CCM, CSM and RoM media compositions can be used. As shown in Figure 2, the transgenic plants of the present invention (OX-820) were taller and had more tillers than the untransformed wild plants. Left experimental panel 1 shows the morphology of whole OX-820 and wild-type (WT) rice plants. Plant A was overexpressing OX-820, which showed increased plant height of the plants. Plant B showed the plant height of the wild type plant Pusa basmati 1 (PB1 ) with a smaller height compared to Ox-820. Right experimental group 2 shows more grain-filled (drooping) ears in OX-820 compared to wild-type (WT) rice plants. Plant C showed more drooping panicles in OX-820 and plant D showed wild type plant Pusabasmati 1 (PB1 ) which had less drooping panicles compared to OX-820. Furthermore, as shown in Figure 3, the transgenic plants exhibited a large number of branches and an increase in ear length. Experimental group 1 showed that plant A overexpressing OX-820 had increased panicle length, and plant B was WT Pusa basmati 1 (PB1 ) with shorter panicle length compared to OX-820. Experimental group 2 showed that plant C OX-820 had more ear branches and plant D wild type (WT) showed less ear branches. Experimental group 3 showed that plant E OX-820 had more kernels, and plant F wild type (WT) showed a lower number of kernels compared to OX-820.

advantage

Overexpression of miR820 in Oryza species as food has been widely used in the agricultural industry as it promotes sustainable agriculture and food quality in many Oryza growing countries. Improved agronomic traits (such as higher yield per acre, plant and panicle vigor, larger tillers and improved plant height, improved tolerance to abiotic stresses, improved ability to use nutrients such as nitrogen, phosphate, and others, and improved harvesting, storage and processing quality) have greatly enhanced the cultivation of Oryza species in many countries.

The following non-limiting examples illustrate some aspects of the invention. All embodiments that are readily conceivable to those skilled in the art are considered to fall within the scope of the present invention, whether or not illustrated by the examples.

Example

The following examples illustrate the construction of plasmids for the transfer of recombinant DNA into plant cells that can be regenerated into transgenic plants of the invention.

i) Selection and growth of plants under optimal conditions: Mature husks were treated with 70% ethanol for 1 minute followed by 10% sodium hypochlorite with Tween-20 drops for 30 minutes under constant slow stirring Rice seeds were surface sterilized. The seeds were repeatedly washed thoroughly with sterile distilled water and blotted dry.

ii) Design of the plasmid construct: The construct was constructed from the backbone of Osa-miR528, a precursor of miRNAs conserved in Oryza species, in vector pRT101 to generate the artificial precursor miR820. Primers specific to the mature sequence of Osa-miR820 (see Table 2) were designed using Web Micro RNA Designer 3, and used to replace miR528 with Osa-miR820. Different primer combinations were used to generate three sets of PCR fragments. These fragments were gel purified and purified by using Sequence No. 3 (Sequence ID No. 3), G3468-: 5'-CTGCAAGGCGATTAAGTTGGGTAACG-3' and Sequence No. 4 (Sequence ID No. 4), G3469-: 5' GCGGATAACAAT TTCACACAGGAAACAG 3 Overlap PCR of 'Universal Primers was performed for fusion to obtain amplified products.

Table 2

serial number Primer name Sequence (5'-3') 1 GFwd CCGGAATTCCGGCAGCAGCAGCCACAGCAAA 2 GRev CGCGGATCCGCGGCTGCTGATGCTGATGCCAT 3 G4368 CTGCAAGGCGATTAAGTTGGGTAACG 4 G4369 GCGGATAACAAT TTCACACAGGAAACAG 5 wxya CAGGTCAACATGGTGGAGCA 6 pRT GTCACTGGATTTTGGTTTTAGG 7 820-Fwd TCGGCCTCGTGGATGG 8 820-Rev GTGCAGGGTCCGAGGT 9 Hyg-Fwd TTCAGCTTCGATGTAGGAGG 10 Hyg-Rev AGAAGAAGATGTTGGCGACC 11 18S_Fwd CTACGTCCCTGCCCTTTGTACA 12 18S_Rev ACACTTCACCGGACCATTCAA

iii) Cloning and expression of Osa-miR820: Plant cells were transformed by Agrobacterium-mediated gene transfer. Agrobacterium strains can be used in pCAMBIA1300 for transformation. The pCAMBIA1300 vector was used for expression in the present invention. These vectors have an enhanced CaMV35S promoter and a CaMV35S poly A signal for termination. The first digit (digit) of the plasmid indicates the presence of the antibiotic hygromycin and the second digit indicates the presence of the antibiotic kanamycin. The third position indicates polylinker pUC18.

Cloning of Osa-miR820 into the Agrobacterium tumefaciens binary vector pCAMBIA1300 was achieved by cloning the amplified PCR product into pRT101 under the CaMV-35S promoter using EcoRI and BamHI sites for directional cloning. Positive clones were subsequently confirmed by double digestion with EcoRI/BamHI. The entire cassette was excised from pRT101-Osa-miR820 by restriction digestion with HindIII and transformed into the pCAMBIA1300 binary vector using the same restriction enzymes. Conversion was confirmed by PCR analysis and restriction digest using SEQ ID No: 7-:5'-TCGGCCTCGTGGATGG-3' and SEQ ID No.8:-5'-GTGCAGGGTCCGAGGT-3'. A recombination cassette contacting pCAMB1300-Osa-miR820 was obtained. Please refer to Figure 1.

插入的OSA-miR820的序列是第13号序列(Sequence No.13)：5-TCGGCCTCGTGGATGGACCAG-3'，并且构建的重组构建体是第14号序列(Sequence No.14)：5'-GCAGCAGCCACAGCAAAATTTGGTTTGGGATAGGTAGGTGTTATGTTAGGTCTGGTTTTTTGGCTGTAGCAGCAGCAGTCGGCCTCGTGGATGGACCAGCAGGAGATTCAGTTTGAAGCTGGACTTCACTTTTGCCTCTCTCTGGTGCATGCACGAGGCCGATTCCTGCTGCTAGGCTGTTCTGTGGAAGTTTGCAGAGTTTATATTATGGGTTTAATCGTCCATGGCATCAGCATCA-3 '.

iv) Agro-infiltration: Agro-infiltration was achieved by pressure infiltration.

Agrobacterium cultures were grown overnight in Lunia broth (LB) medium with appropriate antibiotics and 20 μΜ acetosyringone. Cells were pelleted by brief centrifugation and resuspended in minimal medium (MMA) medium (MS salts, 10 mMMES, pH 5.6, 200 μΜ acetosyringone). Cells were incubated at 28°C for at least 1 hour and then diluted in MES buffer to obtain an OD600 of approximately 0.3-0.4. Infiltrate the homogeneous culture mixture into young leaves with the help of a needle-free syringe by creating a vacuum with the help of fingers on the backside of the leaf and the mouth of the syringe on the ventral side. After 3, 5, 7 and 10 days of infiltration (days post infiltration (dpi)), the infiltrated areas were analyzed for miR820 expression.

v) RNA Isolation: Total RNA was manually extracted from various Oryza species by the guanidinium isothiocyanate protocol. Plant tissue was homogenized in liquid nitrogen and GITC buffer was added along with phenol and chloroform. The mixture was allowed to thaw slowly and centrifuged at high speed (approximately 13000 rpm) for 15 minutes. The aqueous phase was obtained by extraction twice with phenol:chloroform solution. Save the aqueous phase for precipitation with ethanol. The RNA pellet was washed twice with 70% DEPC-ethanol at 13000 rpm for 15 min each and dried at room temperature. The dried RNA pellet was dissolved in DEPC-treated water and stored at -20 °C.

vi) Confirmation of overexpressed transformed lines: Genomic DNA was extracted from transgenic rice lines and wild rice lines. 1 gm of plant material was crushed into liquid nitrogen and homogenized in CTAB buffer. The homogenized samples were kept at 65°C for 30 minutes. The mixture was centrifuged at 10000 rpm for 5 minutes, and the supernatant was collected in a new tube, an equal volume of chloroform:isoamyl alcohol (24:1) was added and centrifuged again. The final pellet was dried at room temperature and dissolved in TE buffer. PCR analysis was performed and amplification products were detected under 2% agarose amplification. Plant material was crushed and homogenized. The mixture was centrifuged and the supernatant collected. Genomic DNA was precipitated and the pellet was incubated. The final precipitate was washed with alcohol, dried and dissolved in buffer. PCR was performed followed by blotting to confirm the expression of the positive rice lines. Blots were performed on HypondN membranes to examine 20 μg of genomic DNA from PCR-positive rice lines digested with KpnI. The hygromycin gene was used as a probe, and hybridization was performed at 62°C for 12 hours. It is then washed and imaged on X-ray film.

As described herein, the plants of the invention are taller compared to wild type rice plants. The plants are shown in Figure 2 and the results are listed in Table 3. Table 3 is a comparison of the biomass associated with overexpressed rice plants containing OX-820. Plant height was measured 95-110 days after sowing. Plant vigor and tiller increase were used as the basis for determining plant height. In this table, the p-test value results show significant differences in biomass-related parameters such as primary tiller number, leaf length, mature plant height, plant biomass dry weight, mature root length, and mature root dry weight. PB-1, PB-2 and PB-3 are wild type Pusa basmati 1 plants (T0), and LI, L2, L3 and L4 are OX-820 transgenic plants (T1). Three independent transgenic events per line are being tested. A total of 15 T1 events were detected. All biomass-related parameters show a significant increase marked by *, as indicated by the resulting p-values in the table.

table 3

Comparison of biomass-related traits in OX-820 and WT

Strain number PB-1 PB-2 PB-3 L1 L2 L3 L4 significant p-value number of primary tillers 6 5 5 8 7 8 8 * 0.007763 Ye Chang 45 42.8 44.2 59.5 56.7 56 54 * 0.000426 mature plant height 34.5 33.7 31.9 37.4 39.6 38.8 38.3 * 0.00642 plant biomass dry weight 26 25.4 25.8 21.5 18 23.5 22.2 * 0.042926 mature root length 25.5 23.6 24.7 22.2 18.4 twenty one 19.5 * 0.031234 mature root dry weight 4.38 4 4.25 4.7 5.02 4.8 4.28 * 0.012547

Transformed rice lines with OX-820 were compared to wild-type rice plants, with particular reference to ear characteristics. The plants are shown in Figure 3 and the results are listed in Table 4 below. Plant vigor has also been used as a parameter to assess ear vigor and is measured as the number of ears per plant and the number and weight of kernels per plant. In this table, p-test value results are shown for agronomically relevant parameters (such as total number of ears, number of spikelets/number of ears, total number of grains/plant, ear length, primary branches/ear, seed weight, grain length and significant difference in grain width). PB-1, PB-2 and PB-3 are wild type Pusa basmati1 plants (T0), and LI, L2, L3 and L4 are OX-820 transgenic plants (T1). Three independent transgenic events per line are being tested. A total of 15 T1 events were detected. All biomass-related parameters show a significant increase marked by *, as indicated by the resulting p-values in the table.

Table 4

Comparison of agronomic traits in OX-820 and WT

Strain number PB-1 PB-2 PB-3 L1 L2 L3 L4 significant p-value Total number of spikes 6 5 6 9 8 9 8 * 0.003126 Number of Spikelets/Number of Spikes 136 131 128 159 168 171 179 * 0.001327 Total number of grains/plant 780 647 804 1104 1206 1373 1232 * 0.00634 ear length 24.1 24.9 23.7 28.2 29.5 26.4 29.8 * 0.003106 1°branch/ear 6 5 6 5 8 7 8 0.348641 1000 - seed weight 20.9 19.8 20.2 23.4 23.2 22.6 21.08 * 0.002323 grain length 10 10.1 10.9 11 11.65 10.8 11.23 0.100159 grain width 2.1 2.2 2.2 2.1 2.4 2.32 2.26 0.327406

vii) Callus induction-: Surface sterilized seeds of rice cv. Incubate on induction medium (CIM). Callus induction medium (CIM) favored the development of the scutellar region into a highly regenerative callus over a period of 3 weeks. Calli were excised and subcultured on fresh callus induction medium (CIM) for 7 days in the dark. For co-infection, the subcultured calli were immersed in the Agrobacterium suspension for 35 min under continuous slow shaking. After infection, calli were blotted dry on sterile filter paper and then incubated for two days at 26°C-28°C in the dark on filter paper moistened with co-cultivation medium (CCM). After co-cultivation, the calli were washed twice for 30 minutes each with high-pressure distilled water containing carbenicillin and cefotaxime (250 mg/ml each). To dilute the toxic effect of antibiotics, a final wash was provided by N6 liquid medium (without any sugar) for 5-7 minutes, and then kept on recovery medium (RM) for 7-10 days to restore the proliferative capacity. Thereafter, calli were washed and dried on sterile filter paper, and cultured on callus selection medium (CSM) in the dark to select for transgenic calli. After 20 days of the first round of selection, the brown or black callus was discarded and the white callus was transferred to fresh CSM medium for a second selection cycle lasting 15 days. This step allows the proliferation of the mini-callus and when the mini-callus begins to grow on the parent callus, each mini-callus is gently detached from the parent callus and transferred to a fresh CSM culture. The third selection lasted 15 days in the base. Select healthy callus for regeneration. After the third selection, healthy calli were transferred to specific regeneration media (RM1-4) and incubated in the dark in a culture room for 7 days. They were then transferred to fresh regeneration medium and incubated at 26°C–28°C under light conditions. After 1-2 weeks, green shoots are seen emerging from the callus. Green shoots developed into shoots and were transferred to rooting medium (RoM) in the presence of hygromycin (50 mg/l) in the light for 20 days. The whole plants were transferred to vermiculite pots, then to soil pots, and grown in the greenhouse. The CIM, CCM, CSM and RoM medium compositions used are detailed below. These media compositions are suitable with some modifications. CIM: MS salts with vitamin B5 + 30g/l sucrose + 0.3g casein hydrolyzate + 2.0mg/l 2,4-D + 0.5g proline + 0.3% vegetable gel, pH 5.8; CCM: liquid N6 Medium+3mg/ml 2,4-D+100μM acetosyringone, pH 5.2; RM: CIM without antibiotic selection; CSM: CIM+250 cefotaxime+50mg/l hygromycin; RM1: vitamin B5 MS salt + 500mg proline + 0.3g casein hydrolyzate + 30g/l sorbitol + 1.0mg/l BAP + 2.0mg/l kinetin + 0.5mg/l NAA, pH5.8; RoM: with vitamin B5 MS salt + 30g sucrose + 3g/l vegetable gel, pH 5.8.

sequence listing

<110> International Center for Genetic Engineering and Biotechnology

<120> Improved transgenic rice plant

<130> PCT0036

<150> 361/DEL/2015

<151> 2015-02-10

<160> 14

<170> PatentIn version 3.5

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<211> 31

<212>DNA

<213> Artificial sequence

<223> oligonucleotide primers

<400> 1

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<210> 2

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<212>DNA

<213> Artificial sequence

<223> oligonucleotide primers

<400> 2

cgcggatccg cggctgctga tgctgatgcc at 32

<210> 3

<211> 26

<212>DNA

<213> Artificial sequence

<223> oligonucleotide primers

<400> 3

ctgcaaggcg attaagttgg gtaacg 26

<210> 4

<211> 28

<212>DNA

<213> Artificial sequence

<223> oligonucleotide primers

<400> 4

gcggataaca atttcacaca ggaaacag 28

<210> 5

<211> 20

<212>DNA

<213> Artificial sequence

<223> oligonucleotide primers

<400> 5

caggtcaaca tggtggagca 20

<210> 6

<211> 22

<212>DNA

<213> Artificial sequence

<223> oligonucleotide primers

<400> 6

gtcactggat tttggtttta gg 22

<210> 7

<211> 16

<212>DNA

<213> Artificial sequence

<223> oligonucleotide primers

<400> 7

tcggcctcgt ggatgg 16

<210> 8

<211> 16

<212>DNA

<213> Artificial sequence

<223> oligonucleotide primers

<400> 8

gtgcagggtc cgaggt 16

<210> 9

<211> 20

<212>DNA

<213> Artificial sequence

<223> oligonucleotide primers

<400> 9

ttcagcttcg atgtaggagg 20

<210> 10

<211> 20

<212>DNA

<213> Artificial sequence

<223> oligonucleotide primers

<400> 10

agaagaagat gttggcgacc 20

<210> 11

<211> 22

<212>DNA

<213> Artificial sequence

<223> oligonucleotide primers

<400> 11

ctacgtccct gccctttgta ca 22

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<211> 21

<212>DNA

<213> Artificial sequence

<223> oligonucleotide primers

<400> 12

acacttcacc ggaccattca a 21

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<212>DNA

<213> Artificial sequence

<223> Synthetic sequence

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tcggcctcgt ggatggacca g 21

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<212>DNA

<213> Artificial sequence

<223> Synthetic sequence

<400> 14

gcagcagcca cagcaaaatt tggtttggga taggtaggtg ttatgttagg tctggttttt 60

tggctgtagc agcagcagtc ggcctcgtgg atggaccagc aggagattca gtttgaagct 120

ggacttcact tttgcctctc tctggtgcat gcacgaggcc gattcctgct gctaggctgt 180

tctgtggaag tttgcagagt ttatattatg ggtttaatcg tccatggcat cagcatca 238

Claims (9)

1. a kind of transgenic rice plant cell, contains recombinant DNA molecules in its genome, the DNA molecular includes coding Osa-miR820 and the DNA sequence dna being operably connected on just direction with promoter；Wherein described plant performance goes out resistance to Salt stress, plant height increase, tiller number increase and Grain Yield increase.

2. a kind of be used to increase the methods of plant products, it include with encode osa-miR820 on just direction with promoter The step of DNA sequence dna being operably connected is to convert plant cell.

3. a kind of be used to increase the method for plant vigor and agronomic characteristics in Oryza species, including by being expressed in expression vector SEQ ID NO.13 nucleotide fragments and over-express miR820 DNA construct.

4. nucleotide fragments according to claim 1, wherein, the nucleotide fragments are Osa-miR820 ripe sequences Row.

5. the rice of the overexpression comprising Osa-miR820, wherein, the basic element of cell division used in culture medium:The ratio of auxin For 6:1.

6. according to the method for claim 1, wherein, the plant comes from grass family, it is preferred from belonging to Oryza.

7. according to the method for claim 1, wherein, the plant comes from India's native species as rice variety as being used as Pusa Basmati 1.

8. the claimed construct being used to prepare in the method for the plant with overexpression, wherein, the construct bag Contain,

A) Osa-miR820 mature sequence；

B) one or more control sequences of the expression of the sequence can be driven；

C) transcription terminator sequences；

D) continuous promoter.

9. miRNA purposes is over-expressed in described rice plant in claim 1, by promoting the Oryza under stress conditions The vigor of plant and fringe in species, tiller and cause output increased.