CN106282201A - Maize Transcription Factor ZmbHLH2 and application thereof - Google Patents

Abstract

The present invention relates to maize transcription factor ZmbHLH2 and its application. The nucleotide sequence of the maize transcription factor ZmbHLH2 provided by the present invention is shown in SEQ ID NO.1, and the transcription factor is related to maize drought resistance. By transforming Arabidopsis thaliana with ZmbHLH2 gene, the drought resistance function of the gene was verified, and it was proved that the tolerance ability of Arabidopsis transgenic ZmbHLH2 gene to drought stress was improved. The gene can be used in drought-resistant transgenic research of maize and other crops. The maize transcription factor ZmbHLH2 gene of the invention is from maize, does not generate gene drift, and provides new gene resources for improving plant drought resistance.

Description

Maize Transcription Factor ZmbHLH2 and Its Application

technical field

The invention relates to the technical field of genetic engineering, in particular to the maize transcription factor ZmbHLH2 gene and its application.

Background technique

Maize cultivation is often threatened by drought, which affects maize photosynthetic and physiological activities, grain formation and yield. In view of the important role of maize in food security, the drought resistance of maize has been studied from the aspects of population genetic improvement, cultivation measures and physiological and biochemical levels. At the population level, nine drought-resistant inbred lines were screened by studying the total root length (TRL) and root surface area (TSA) of maize inbred lines closely related to drought resistance. A study of 13 drought-resistant populations found that there was a significant positive correlation between palmitic acid and oleic acid in drought-resistant maize varieties. At the cultivation level, it was found that spraying GABA, reducing lateral root density and increasing terpene synthesis could improve the drought resistance of maize.

The development of molecular biology, especially the completion of the complete genome sequencing of maize provides strong support for the genetic improvement of drought resistance in maize. For example, transforming microbial cold shock protein CspB gene can effectively improve the drought resistance of maize variety MON87460 at silking and heading stages; overexpressing ZmARGOS1 and ZmARGOS8 genes can improve the drought resistance of Arabidopsis and maize by reducing the sensitivity to ethylene. At the post-translational level, alternative splicing of the genome can also improve drought tolerance in maize.

Transcription factors are widely used in the study of plant drought resistance because they can regulate multiple genes at the same time. Transcription factor families such as CCAAT, homeodomain, bHLH, NAC, AP2/ERF, bZIP and WRKY all have the potential to regulate drought resistance. For example, the maize HD-Zip gene Zmhdz10 positively regulates the resistance to drought and salt stress in rice and Arabidopsis. NAC transcription factors are key transcription factors of drought resistance in maize, and 24 NAC transcription factors may be related to drought resistance of maize. Maize ZmbZIP72 can improve the resistance of Arabidopsis to drought and salt stress. Therefore, identifying the functions of members of the maize transcription factor family will help to screen drought-resistant genes in maize and provide assistance for molecular marker-assisted breeding.

The bHLH transcription factor is the second largest type of transcription factor in plants. It contains a highly conserved bHLH domain, which contains about 60 amino acids and generally consists of two functionally completely different regions: the basic region and the helix-loop-helix structure area. The N-terminal basic region functions as a DNA binding domain and consists of about 15 amino acid residues, including about 6 basic amino acid residues. It can specifically bind to the cis-acting element E-box (5'-CANNTG-3') or G-box (5'-CACGTG-3') in the promoter region of the target gene. The helix-loop-helix region at the C-terminus is responsible for the formation of homologous or heterodimers, and bHLH family members mainly function in the form of dimers. According to the similarity of bHLH domain sequences, a total of 638 bHLH genes from Arabidopsis, poplar, rice, moss and 5 algae genomes could be divided into 32 subfamilies. In the study of ABA signaling pathway, it was found that the bHLH transcription factor AtMYC2 and the MYB transcription factor AtMYB2 are transcriptional activators of ABA signaling, but they are repressors in the light growth and development of plants regulated by blue light.

It was subsequently found that the driving function of bHLH transcription factors can be divided into two pathways: ABA-dependent and ABA-independent. Studies of members of the plant bHLH family have shown that a second subgroup of bHLH transcription factor functions is associated with injury, drought, oxidative stress, jasmonic acid, and abscisic acid signaling. It has been proved that bHLH transcription factors are usually combined with other transcription factors and proteins to regulate plant growth and development, hormone signal transduction and stress response processes. The R protein of maize is the first bHLH transcription factor identified in plants, and together with the R2R3-MYB transcription factor C1, activates the expression of anthocyanin synthesis genes. BA1 is a bHLH transcription factor required for maize axillary meristem formation. The bHLH transcription factor ZmPTF1 regulates carbon metabolism and root growth in maize. Overexpression of ZmPTF1 in maize can improve plant tolerance to low phosphorus stress. bHLH transcription factors play an important role in the response of plants to abiotic stress. It has been proved that bHLH is involved in the drought resistance response of tobacco and Populus euphratica. However, little is known about the function of maize bHLH transcription factors in stress response.

Contents of the invention

The object of the present invention is to provide maize transcription factor ZmbHLH2 gene and its encoded protein.

Another object of the present invention is to provide the application of the maize transcription factor ZmbHLH2 gene in improving the drought resistance ability of plants.

In order to achieve the purpose of the present invention, the present invention clones the corn transcription factor ZmbHLH2 gene from the leaves of the corn variety Dongnong 255 cultivated by the Agricultural College of Northeast Agricultural University, and the cDNA sequence of the ZmbHLH2 gene is:

i) the nucleotide sequence shown in SEQ ID NO.1; or

ii) A nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO.1 is substituted, deleted and/or increased by one or more nucleotides and expresses the same functional protein; or

iii) a nucleotide sequence that hybridizes to the sequence shown in SEQ ID NO.1 and expresses the same functional protein under stringent conditions, and the stringent conditions are 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS solution, hybridize at 65°C, and wash the membrane with this solution; or

iv) A nucleotide sequence having more than 90% homology with the nucleotide sequence of i), ii) or iii) and expressing the same functional protein.

The sequencing results showed that the maize transcription factor ZmbHLH2 is a bHLH family transcription factor, and the open reading frame of the ZmbHLH2 gene is 2118bp in full length, encoding a protein (SEQ ID NO.2) consisting of 7055 amino acids. Based on the bioinformatics analysis of the ZmbHLH2 gene and the expression pattern judgment under drought and other stresses, the gene may be involved in the stress resistance pathway of maize and other plants.

The invention also provides the biological material containing the ZmbHLH2 gene of the maize transcription factor, and the biological material is an expression cassette, a vector, an engineering bacterium or a cell.

The expression vector carrying the gene of interest can be introduced into plant cells by conventional biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press , New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology, ^2nd Edition).

The present invention also provides the application of the maize transcription factor ZmbHLH2 gene in improving the drought resistance ability of plants (for example, Arabidopsis, maize, etc.).

The invention also provides the application of the maize transcription factor ZmbHLH2 gene in improving plant germplasm resources.

The invention also provides the application of the maize transcription factor ZmbHLH2 gene in preparing drought-resistant transgenic plants.

The present invention also provides a method for constructing transgenic plants, comprising the following steps:

(1) extract the total RNA in the corn leaves, and reverse transcribe to obtain the first strand of cDNA;

(2) Using the first strand of DNA as a template and ZmbHLH2F and ZmbHLH2R as primers, the cDNA sequence of the transcription factor ZmbHLH2 gene was obtained by PCR amplification reaction, and its cDNA fragment was ligated into the pMD18-T vector and transformed into E. coli DH5α strain Cloning and sequencing verification were carried out, and the correct plasmid was named ZmbHLH2-18T after sequencing verification;

(3) Using the ZmbHLH2-18T plasmid as a template, ZmbHLH2-ORF-F: 5′-CG GAATTC ATGAACCTGTGGACG-3′ (underlined is the EcoR I restriction site) and ZmbHLH2-ORF-R: 5′-CG GGATCC CCTGCCCATGGCAGA- 3' (the underline is the BamHI restriction site) is the primer to amplify the ORF of the ZmbHLH2 gene, and the amplified product is digested by EcoR I and BamHI and then connected to the expression vector after the same digestion. Agrobacterium GV3101 strain was transformed with the plasmid of ZmbHLH2 gene ORF;

(4) Using transgenic technology to transform the target plant with the Agrobacterium carrying the transformation plasmid to obtain a transgenic stable genetic plant.

Wherein, the sequences of primers ZmbHLH2F and ZmbHLH2R described in step (2) are as follows:

ZmbHLH2F: 5′-ATCCGCATTCTGAACCATT-3′

ZmbHLH2R: 5'-TGCCTATCGTGTTGTAACC-3'.

The present invention further provides fluorescent quantitative PCR detection primers of maize transcription factor ZmbHLH2 gene, and the primer sequences are as follows:

ZmbHLH2-QF1: 5′-GACGCCATCCTCCTACATC-3′

ZmbHLH2-QR1: 5'-GCTCTCCTCCTTGATTACC-3'.

The expression of ZmbHLH2 gene in the drought-resistant and non-drought-resistant inbred lines of maize and after the treatments of drought, jasmonic acid, abscisic acid and hydrogen peroxide were detected. The results showed that the ZmbHLH2 gene was affected by drought, jasmonic acid, abscisic Acid and hydrogen peroxide treatment induce expression.

In the present invention, the ZmbHLH2 gene is transformed into Arabidopsis thaliana to carry out the function research of the gene, and it is verified that the tolerance ability of the transgenic Arabidopsis thaliana is improved to drought stress. The gene can be used in drought-resistant transgenic research of maize and other crops. The transcription factor gene ZmbHLH2 related to maize drought resistance in the invention is from maize, does not generate gene drift, has little impact on the environment, and provides new gene resources for improving plant drought resistance.

Description of drawings

Fig. 1 is the amino acid sequence homology alignment result of the conserved region of the ZmbHLH2 transcription factor in Example 1 of the present invention. The sequences of bHLH proteins of other species are pea PsbHLH, protein number ADO13282.1[Pisum sativum]; potato StbHLH, protein number CDJ79759.1[Solanum tuberosum]; Arabidopsis AtbHLH4, protein number O49687.1[Arabidopsis]; Arabidopsis Mustard AtbHLH5, protein number Q9FIP9.1 [Arabidopsis].

Fig. 2 shows the expression of ZmbHLH2 in drought-resistant inbred lines and non-drought-resistant inbred lines detected by fluorescent quantitative PCR in Example 2 of the present invention. Drought-resistant inbred lines: Zheng 58, Ji 853, Qi 319, Han 21, X178, 7922, Ye 52106 and K12; drought-sensitive inbred lines: Dong 91, Dong 46, Dong 26-6, Dong 34, red corn and PH4CV.

Figure 3 shows the expression of ZmbHLH2 gene in Dongnong 255 maize leaves treated with drought, jasmonic acid, abscisic acid and hydrogen peroxide.

Fig. 4 shows that the drought tolerance of the ZmbHLH2 transgenic Arabidopsis line in the soil is significantly improved in Example 1 of the present invention.

detailed description

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.

Unless otherwise specified, the reagents, consumables, and biological materials used in the embodiments of the present invention are all commercially available.

Cloning of Example 1 Maize Transcription Factor ZmbHLH2 Gene

The maize transcription factor ZmbHLH2 gene was cloned from the leaves of the maize variety Dongnong 255 (this maize variety has been approved and promoted by relevant departments) cultivated by the College of Agriculture of Northeast Agricultural University by RT-PCR technology. The specific method is as follows:

1. Extract the total RNA from the maize leaves, and reverse transcribe to obtain the first strand of cDNA.

2. The cDNA sequence of the transcription factor ZmbHLH2 gene was obtained by PCR amplification reaction using the first strand of DNA as a template and ZmbHLH2F and ZmbHLH2R as primers.

Wherein, the sequences of primers ZmbHLH2F and ZmbHLH2R described in step (2) are as follows:

ZmbHLH2F: 5′-ATCCGCATTCTGAACCATT-3′

ZmbHLH2R: 5'-TGCCTATCGTGTTGTAACC-3'.

Use 2×Taq PCR MasterMix (Tiangen Biochemical) for PCR amplification. The reaction system is: 2 μL of cDNA template, 1 μL of primers ZmbHLH2F and ZmbHLH2R (10 μmol/L), 25 μL of 2×PCR MasterMix, and add ddH ₂ O to 50 μL.

The PCR amplification program was: denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 50 s, and extension at 72°C for 2 min, a total of 35 cycles; finally, extension at 72°C for 10 min.

3. After the amplified product was subjected to 1% agarose gel electrophoresis, the target fragment was excised, recovered and purified by gel, cloned into the pMD19-T vector, detected by colony PCR, and sent to Shanghai Lifei Biotechnology Co., Ltd. for sequencing verification. Sequencing results showed that the maize transcription factor ZmbHLH2 belongs to the bHLH family of transcription factors, the open reading frame of the ZmbHLH2 gene (SEQ ID NO.1) is 2118bp in full length, and encodes a protein ZmbHLH2 (SEQ ID NO.2) consisting of 705 amino acids. The amino acid sequence homology alignment results of the conserved regions of ZmbHLH2 transcription factors are shown in Figure 1.

Expression analysis of embodiment 2 ZmbHLH2 gene when subjected to stresses such as drought

The expression pattern of ZmbHLH2 gene in different drought-resistant inbred lines was detected by fluorescent quantitative PCR. Drought-resistant inbred lines, Zheng 58, Ji 853, Qi 319, Han 21, X178, 7922, Ye 52106 and K12; drought-sensitive inbred lines, Dong 91, Dong 46, Dong 26-6, Dong 34, red corn and PH4CV (See Figure 2), wherein the expression of ZmbHLH in drought-resistant inbred lines was significantly higher than that in non-drought-resistant inbred lines.

The following fluorescent quantitative PCR detection primers were designed according to the ZmbHLH2 gene sequence:

ZmbHLH2-QF1: 5′-GACGCCATCCTCCTACATC-3′

ZmbHLH2-QR1: 5′-GCTCTCCTCCTTGATTACC-3′

ZmACTIN-F: 5′-ACCTCACCGACCACCTAATG-3′

ZmACTIN-R: 5′-GCAGTCTCCAGCTCCTGTTC-3′

ZmACTIN (GenBank accession number: GRMZM2G126010) was used as an internal reference gene. The CFX-96 fluorescent quantitative PCR instrument of BioRad Company was used for fluorescent quantitative PCR, and the reaction program was: 95°C pre-denaturation for 30s; 94°C for 5s, 60°C for 20s, 72°C for 20s, and 45 cycles. After the amplification, the melting curve was drawn, and the temperature was gradually raised from 50°C to 95°C at a heating rate of 0.2°C/s, and the fluorescence signal was detected during the whole process. The expression level of the ZmbHLH2 gene is calculated using the formula Qt=2-Ct(ZmACTIN)-Ct(ZmbHLH2), where Ct represents the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold value.

The results of real-time quantitative PCR detection showed that Dongnong 255 maize seedlings at the 3-leaf stage were subjected to drought (continuous water cut-off for 7 days), 200 μmol L ^-1 (w/v) jasmonic acid, 200 μmol L ^-1 (w/v) abscisic acid and 2 After % (v/v) hydrogen peroxide treatment, the expression of ZmbHLH2 gene was induced ( FIG. 3 ). After one day of drought treatment, the expression level of the transcription factor ZmbHLH2 increased by 6.31 times compared with the control, and then the expression level decreased, but the expression levels were 3.09, 4.13, 3.66, 1.79 and 2.84 times of the control at 3-7 days of drought, respectively. After jasmonic acid treatment, the expression levels were the highest at 6 and 10 hours, which were 7.15 and 10.77 times that of the control, respectively. The expression level of abscisic acid treatment was the highest at 48h, which was 25.52 times that of the control. The expression level of hydrogen peroxide treatment was the highest at 6h and 10h, which was 31.49 times better than that of the control (41.02). It indicated that ZmbHLH2 was a drought-responsive gene, and its expression was significantly up-regulated by the above four treatments.

Example 3 Obtaining and Drought Resistance Verification of ZmbHLH2 Gene-transferred Arabidopsis Plants

Total RNA was extracted from maize leaves, and the first strand of cDNA was obtained by reverse transcription. The cDNA sequence of the transcription factor ZmbHLH2 gene was obtained by PCR amplification reaction using the first strand of DNA as a template and ZmbHLH2F and ZmbHLH2R as primers, and its cDNA fragment was ligated into the pMD18-T vector and transformed into Escherichia coli DH5α strain for cloning and Sequencing verification, the correct plasmid was named ZmbHLH2-18T; with the ZmbHLH2-18T plasmid as a template, ZmbHLH2-ORF-F: 5'-CG GAATTC ATGAACCTGTGGACG-3' (the underline is the EcoR I restriction site) and ZmbHLH2- ORF-R: 5′-CG GGATCC CCTGCCCATGGCAGA-3′ (the underline is the BamHI restriction site) is used as a primer to amplify the ORF of the ZmbHLH2 gene, and the amplified product is double digested by EcoR I and BamHI and connected to The final expression vector was transformed into the Agrobacterium GV3101 strain with the plasmid carrying the ZmbHLH2 gene ORF by electroporation. The present invention introduces the constructed ZmbHLH2 transformation vector into Agrobacterium tumefaciens GV3101 by electric shock method, transforms the ZmbHLH2 gene into wild-type Arabidopsis plants (col) through the Agrobacterium-mediated method, and identifies the transgenic positive seedlings of the target plants , the positive homozygous plants of the transgenic T3 generation of the target plant were screened and stress resistance analysis, as shown in Figure 4, the results showed that the tolerance of the ZmbHLH2 gene Arabidopsis to drought stress was improved, providing a basis for the application of the ZmbHLH2 gene in other plants. good foundation. The drought-resistant gene ZmbHLH2 comes from maize, which does not cause gene drift and has little impact on the environment.

Phenotype after 7 days of drought and water deprivation, compared with wild-type (col) Arabidopsis plants, the drought tolerance of ZmbHLH2 transgenic lines in soil was significantly improved (Fig. 4). The wild-type Arabidopsis all died after drought treatment, and the survival rate was significantly lower than that of transgenic materials.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. Maize Transcription Factor ZmbHLH2, it is characterised in that its cDNA sequence is:

I) nucleotide sequence shown in SEQ ID NO.1；Or

Ii) nucleotide sequence shown in SEQ ID NO.1 is substituted, lacks and/or increases one or more nucleotide and expression The nucleotide sequence of identical function protein；Or

Iii) under strict conditions with sequence hybridization shown in SEQ ID NO.1 and express identical function protein nucleotides sequence Row, described stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C Hybridization, and wash film with this solution；Or

Iv) and i), ii) or nucleotide sequence iii) there is more than 90% homology and express the nucleoside of identical function protein Acid sequence.

2. Maize Transcription Factor ZmbHLH2, it is characterised in that its aminoacid sequence contains the amino as shown in SEQ ID NO.2 Acid sequence.

3. contain the biomaterial of Maize Transcription Factor ZmbHLH2 described in claim 1 or 2, it is characterised in that described biological material Material is expression cassette, carrier, engineering bacteria or cell.

4. the application in improving plant drought ability of the Maize Transcription Factor ZmbHLH2 described in claim 1 or 2.

5. the application in plant germplasm resource is improved of the Maize Transcription Factor ZmbHLH2 described in claim 1 or 2.

6. the application in preparation drought resisting transgenic plant of the Maize Transcription Factor ZmbHLH2 described in claim 1 or 2.

7. the application as described in claim 4-6 is arbitrary, it is characterised in that described plant includes arabidopsis, Semen Maydis.

8. the construction method of a transfer-gen plant, it is characterised in that comprise the following steps:

(1) extracting the total serum IgE in maize leaf, reverse transcription obtains cDNA the Ith chain；

(2) with DNA the Ith chain as template, ZmbHLH2F and ZmbHLH2R is primer, obtains transcription factor by pcr amplification reaction The cDNA sequence of ZmbHLH2 gene；

(3) utilize electric shocking method by the Plastid transformation Agrobacterium with ZmbHLH2 gene cDNA sequence；

(4) utilize transgenic technology by with the Agrobacterium-mediated Transformation target plant converting plasmid, it is thus achieved that transgenic is stablized heredity and planted Strain；

Wherein, the sequence of step (2) described primer ZmbHLH2F and ZmbHLH2R is as follows:

ZmbHLH2F:5 '-ATCCGCATTCTGAACCATT-3 '

ZmbHLH2R:5 '-TGCCTATCGTGTTGTAACC-3 '.

9. for detecting the primer pair of Maize Transcription Factor ZmbHLH2, it is characterised in that primer sequence is as follows:

ZmbHLH2-QF1:5 '-GACGCCATCTCCTACATC-3 '

ZmbHLH2-QR1:5 '-GCTCTCCTCCTTGATTACC-3 '.

10. contain test kit or the detectable of primer pair described in claim 9.