Gene GhUCE for regulating and controlling growth period and drought resistance of crops and application of gene GhUCE

Abstract

The invention belongs to the technical field of cotton gene engineering, and particularly relates to a gene GhUCE for regulating and controlling the growth period and drought resistance of crops and application of the gene GhUCE. The GhUCE gene disclosed by the invention is introduced into upland cotton for overexpression, so that an early flowering and drought-resistant transgenic cotton plant is obtained, and early-maturing and drought-resistant transgenic materials such as wheat, tobacco, arabidopsis thaliana, rice and the like are also obtained. The invention provides a new thought for the early blossoming and drought resisting mechanism of plants, and has important significance in cultivation of early-maturing and drought-resisting crop varieties or research.

Description

Technical Field

[0001] The invention belongs to the technical field of cotton genetic engineering, and in particular relates to a gene GhUCE for regulating crop growth period and drought resistance and an application thereof. Background Art

[0002] As the world's most important natural fiber crop, the precise regulation of flowering time and drought resistance of upland cotton directly determine the coordinated improvement of core agronomic traits such as growth period optimization, fiber quality formation and environmental adaptability. Against the backdrop of intensified global climate change and frequent extreme weather, shortening the growth cycle and cultivating early-maturing varieties have become important strategies to ensure the sustainable development of my country's cotton industry. However, as a typical quantitative trait, the genetic regulatory network of cotton flowering time is complex and its molecular mechanism has not yet been clarified, which seriously restricts the application of molecular design breeding. The applicant previously systematically identified multiple main effect loci that control important agronomic traits of upland cotton, including GhUCE regulating the growth period, through large-scale genome-wide association analysis (GWAS) of natural populations of upland cotton, but the intrinsic relationship between genotype and phenotype has not yet been clarified.

[0003] This study, based on the laboratory's previous GWAS, focused on the ubiquitin-binding enzyme GhUCE, a key candidate gene that regulates flowering time. It also analyzed its application potential in early maturity and drought resistance breeding of different crops, providing a theoretical basis and technical support for the precise improvement of crop flowering time and drought resistance. Summary of the invention

[0004] The purpose of the present invention is to provide a gene GhUCE for regulating crop growth period and drought resistance and its application.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: A GhUCE gene for regulating plant development, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0006] An expression cassette comprising a GhUCE gene.

[0007] A recombinant expression vector is a plant expression vector, which is obtained by connecting a GhUCE gene fragment to a vector Super-1300 or Cas9 to obtain recombinant plasmids Super-1300-GhUCE and Cas9-GhUCE respectively.

[0008] A transgenic cell line comprising a GhUCE gene.

[0009] Furthermore, the present invention provides an application of the GhUCE gene, specifically, an application in regulating crop growth period and drought resistance; the GhUCE gene is introduced into target crops and overexpressed to obtain early-maturing and drought-resistant transgenic plants, wherein early maturation specifically refers to early flowering and early heading. The plants are sea island cotton, Asian cotton, upland cotton, tobacco, Arabidopsis, rice, wheat, etc.

[0010] The present invention also provides a method for cultivating transgenic plants, comprising the following steps: introducing a coding gene GhUCE into a recipient plant to obtain a transgenic plant whose growth and development are different from those of the recipient plant. In the above method, the introduction of the gene GhUCE into the recipient plant can be achieved by introducing a recombinant plasmid into the recipient plant, and the recombinant plasmid is Super-1300-GhUCE.

[0011] A person skilled in the art can adopt known methods to mutate the nucleotide sequence of the gene GhUCE of the present invention. Those nucleotides obtained after artificial modification and having 80% or higher identity with the nucleotide sequence of the gene GhUCE of the present invention, as long as they encode the gene GhUCE and have the function of the gene GhUCE, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0012] The invention has the advantages that the GhUCE gene of the invention regulates the growth period of plants to advance, promotes early maturity of crops and promotes drought resistance of crops. The invention first uses ClustalX and DNAMAN software to analyze the evolutionary relationship between the GhUCE protein and the homologous protein of the model plant Arabidopsis thaliana, and the homology with the AtUCE protein is 97.30%, indicating that the UCE protein is extremely conservative in plants. The research team previously used qRT-PCR technology to detect the expression amount of GhUCE in the cotton stem tip that determines the key node of the flowering period in two haplotypes, showing that the expression amount of GhUCE in the early-flowering material is higher than that in the late-flowering material, and it is speculated that GhUCE may play an important role in regulating the growth period of cotton. Through cotton overexpression and knockout experiments, it is verified that the GhUCE gene regulates the growth period of cotton. Next, the GhUCE overexpression vector is transferred into Arabidopsis thaliana, wheat, tobacco, rice, etc., and it is found that the growth period of the above crops and model plants is significantly advanced; at the same time, it is verified that the overexpression of GhUCE in cotton, wheat, and rice also leads to extreme drought resistance of crops, indicating that GhUCE not only promotes early flowering of crops but also promotes drought resistance.

[0013] The present invention provides the application of GhUCE gene in regulating the growth period of cotton. After the GhUCE gene of the present invention is transferred into crops, the growth period of cotton and other crops can be significantly advanced and drought-resistant, which is of great significance for research and improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1Functional conservation analysis of UCE genes in different species (a) Genetic complementation experiment of UCE homologous genes in different species in Arabidopsis Atuce mutant. At, Arabidopsis; Os, rice; Zm, maize; Ta, wheat; Gm, soybean. Scale bar is 1 cm. (b) GUS staining of transgenic Arabidopsis flower buds in (a). (c) Expression level of GUS gene in transgenic Arabidopsis flower bud tissue. (d) GUS activity detection in transgenic Arabidopsis flower bud tissue. Data in (cd) represent mean ± standard deviation (n≥ 3). Significant differences were determined based on one-way analysis of variance (P<0.05).

[0015] Figure 2 Molecular identification of GhUCE overexpression and mutant plants; where: (a) Transcription levels of GhUCE in flower buds of wild-type and overexpression plants, with GhUBQ7 as an internal reference. Data represent mean ± SD (n = 3), asterisks indicate significant differences calculated by T test, ***P < 0.001. (b) Immunoblotting analysis of GhUCE-Flag protein in flower buds of wild-type and GhUCE overexpression plants, with Actin as an internal reference. (c) Identification of editing types of Ghuce mutants based on Sanger sequencing. PAM sites and sgRNA sequences are highlighted with red and green backgrounds, respectively, and nucleotide substitutions or deletions are marked in red.

[0016] Figure 3 Analysis of flowering time phenotypes of GhUCE overexpression and mutant lines (a) Flowering phenotypes of WT, GhUCE-OX and Ghuce mutant plants under LD conditions. (b) Statistical analysis of flowering time of plants in (a). (c) Flowering phenotypes of WT, GhUCE-OX and Ghuce mutant plants under SD conditions. (d) Statistical analysis of flowering time of plants in (c). Scale bar: 10 cm. Data (b, d) represent mean ± SD (n = 10). Significant differences (P < 0.05) were calculated by one-way ANOVA.

[0017] Figure 4 Effect of GhUCE on heading time of wheat (a) Heading phenotype of wild-type and GhUCE overexpressing wheat lines, scale bar is 15 cm. (b) Heading time statistics of wild-type and GhUCE overexpressing wheat lines. Data are mean ± SD (n = 10). Significant differences were calculated by one-way ANOVA (P < 0.05).

[0018] Figure 5 Heading phenotypes of wild-type and GhUCE-overexpressing rice lines.

[0019] Figure 6Flowering phenotypes of wild-type and GhUCE-overexpressing tobacco lines.

[0020] Figure 7 Identification of drought resistance in transgenic lines of cotton, rice, and wheat. DETAILED DESCRIPTION

[0021] In the following examples, the test methods, unless otherwise specified, are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources; quantitative experiments were all repeated three times, and the results were averaged.

[0022] Experimental Example 1: Functional study of UCE genes in different species During the evolutionary process, many genes have undergone conservative evolution, which may have similar or identical functions across species. Given that the GhUCE gene is crucial for early flowering of upland cotton, this study hopes to reveal the conservation of the UCE regulation mechanism of flowering and the functional differences between different species. Arabidopsis, as a classic model plant, is an ideal material for studying the evolutionary mechanism of gene function. The orthologous gene of GhUCE in Arabidopsis is AtUBC10. There are currently no reports on the function of this gene. In order to avoid confusion, this article named AtUBC10 AtUCE. By analyzing the flowering phenotypes of the wild-type Arabidopsis Col-0 and the T-DNA insertion mutant of Atuce, it was found that the functional loss of AtUCE did not delay the flowering time of Arabidopsis ( Figure 1 a). Since the GhUCE promoter has strong activity in Arabidopsis flowers ( Figure 1 b), so the coding sequence of AtUCE was cloned and constructed in tandem with the promoter of GhUCE to the upstream of the pCAMBIA1381-GUS plasmid reporter gene, and genetic complementation experiments were performed by transforming Arabidopsis Atuce mutants. The results showed that there was no significant difference in the flowering time of complemented Arabidopsis, Col-0 and Atuce mutants ( Figure 1 a), indicating that the AtUCE gene of Arabidopsis thaliana is not involved in regulating flowering time. Next, the homologous sequences of GhUCE protein in important crops such as rice, corn, wheat, and soybean were retrieved through the NCBI website, and these genes were genetically complemented in Atuce mutants. It was found that only the GhUCE complemented Arabidopsis plants showed the phenotype of early flowering and reduced rosette leaves, while the transgenic plants of UCE of other species were consistent with Col-0 and Atuce mutants in flowering time ( Figure 1 a). After testing the transcription level of the GUS reporter gene in the flower buds of the complemented strains, it was found that the GUS expression levels in all transgenic strains were consistent ( Figure 1c). We initially thought that the UCE gene might have evolved a flowering-regulating function in upland cotton, so we tested the GUS activity in the flower buds of all transgenic Arabidopsis. Surprisingly, except for the high level of GUS activity detected in Arabidopsis with GhUCE complementation, almost no GUS activity was detected in transgenic Arabidopsis with UCE of other species ( Figure 1 d). Qualitative staining of GUS using X-Gluc showed that obvious GUS signal accumulation could only be observed in the flower buds of GhUCE-complemented Arabidopsis, while almost no blue spots were observed in the flower buds of the other complemented lines ( Figure 1 b), which is completely consistent with the results of GUS activity detection. Based on the above results, it is speculated that the UCE genes of other species can be transcribed normally in Arabidopsis, but after translation, they may be modified at the protein level and thus cannot exist stably.

[0023] Experimental Example 2: Construction of GhUCE gene overexpression vector and knockout vector and verification of its effect on promoting early flowering in cotton After extensive and in-depth research, the inventors discovered for the first time a gene that can regulate early flowering of cotton, GhUCE. In order to study the biological function of GhUCE in upland cotton, the downstream of the GhUCE coding sequence was fused with a Flag tag and constructed into an overexpression vector driven by CaMV 35S. CRI16 was used as the receptor material, and a positive strain (GhUCE-OX) was obtained through Agrobacterium-mediated genetic transformation. Two independent T 2 The overexpression plants were then sequenced for molecular identification. First, total RNA from flower bud tissue was extracted and the transcription level of GhUCE was detected by RT-qPCR. The results showed that the expression of GhUCE in the overexpression plants increased by 60-70 times compared with WT ( Figure 2 a). Then, the total protein of WT and GhUCE-OX flower buds was extracted, and western blot analysis was performed using Flag antibody, and it was found that GhUCE-Flag could normally express the corresponding protein ( Figure 2 b), indicating that the GhUCE fragment driven by CaMV 35S has been integrated into the cotton genome and can be transcribed and translated normally. The CRISPR / Cas9 gene editing system was further used to knock out the GhUCE gene. The specific sgRNA sequence was screened through the online target design platform (http: / / www.rgenome.net / cas-designer / ), the sgRNA was cloned into the gene editing vector, and multiple positive strains were obtained through genetic transformation. After continuous self-pollination of these positive plants, T 2DNA of the first generation plants was used to amplify the target region by PCR using specific primers, and the product was cloned by TA and then sequenced by Sanger sequencing. Two homozygous mutant lines (Ghuce-1 and Ghuce-2) with frameshift mutations in the GhUCE open reading frame were selected for subsequent phenotypic analysis ( Figure 2 c).

[0024] GhUCE T 3 The transgenic lines and mutants were planted in greenhouses under LD and SD conditions, respectively, and the time from the sowing date to the opening of the first flower bud was used as the statistical standard for flowering time. After statistics, it was found that under LD conditions, the flowering time of GhUCE-OX plants was about 10 days earlier than that of WT, while the knockout of GhUCE caused the flowering time of cotton to be delayed by about 25 days ( Figure 3 a / b), indicating that GhUCE has a positive regulatory effect on cotton flowering under LD conditions. Under SD conditions, the flowering time of GhUCE-OX plants was about 20 days earlier than that of WT, while the flowering time of Ghuce mutants was almost the same as that of WT ( Figure 3 c / d), indicating that the functional loss of GhUCE has no effect on flowering time under SD conditions. Combined with the RT-qPCR analysis results in the above experiment ( Figure 2 a) The transcription level of GhUCE in the flower buds of overexpressing plants increased by nearly 60 times, resulting in a large accumulation of its protein, which may promote flowering in a dose-dependent manner under SD conditions. However, this phenomenon does not reflect the universal effect of GhUCE in natural populations, and it is speculated that the endogenous expression pattern driven by GhUCE's own promoter may not have significant flowering regulatory activity under SD conditions.

[0025] Experimental Example 3: GhUCE promotes early heading and other crops such as wheat and rice The above results indicate that the UCE genes of multiple crops do not have the function of promoting flowering in Arabidopsis, which means that cotton UCE is a new gene resource and is expected to be used for the genetic improvement of grain crops. As one of the long-standing grain crops in my country, wheat occupies a staple food status in the northern region, and its consumption exceeds 50% of the total national grain consumption. As a typical long-day plant, extending the light time will shorten the growth period of wheat ears and promote early heading, but this will lead to a decrease in the number of fertile florets and grains per ear. Wheat heading period is an important agronomic trait closely related to flowering time. Arabidopsis complementation experiments showed that TaUCE driven by a specific promoter did not affect bolting and flowering time ( Figure 1a). GhUCE was constructed downstream of the Ubi promoter in the pGA3426 vector, and the wild-type wheat Fielder was used as the recipient material. Agrobacterium carrying Ubi-GhUCE was used to infect the stem tip to obtain two wheat overexpression lines. Under LD conditions, the heading dates of the L1 and L2 lines of Ubi:GhUCE were 13 and 18 days earlier than those of Fielder, respectively ( Figure 4 ). Subsequently, GhUCE was overexpressed in rice and tobacco, and the heading period of rice was also advanced ( Figure 5 ) and the early-flowering phenotype of tobacco ( Figure 6 ), indicating that GhUCE can positively regulate flowering or heading time in multiple crops.

[0026] Experimental Example 4: GhUCE simultaneously promotes drought resistance in other crops such as cotton, wheat and rice After analyzing the genome of the upland cotton population, it was found that the GhUCE-SNPA allele variant originated from a tetraploid wild cotton (G. ekmanianum, (AD)6) growing in extremely arid areas, but only exists in a few modern cultivated upland cotton varieties. It is speculated that it may be the result of artificial selection intervention. Although the GhUCE-SNPA allele has an important contribution to the early flowering of upland cotton, in breeding practice, since fiber quality and yield are the core goals of cotton breeding, the SNPA locus may be gradually eliminated due to its negative impact on fiber length. Many plants have the phenomenon of rapid flowering after the dry season, which is called "drought escape". This is a reproductive strategy for adapting to climate change during plant evolution. Therefore, the SNPA allele variant may be the result of long-term drought domestication. If this speculation is correct, GhUCE should be involved in regulating the drought stress response of cotton. For this reason, GhUCE was subsequently overexpressed in wheat and rice, and drought tolerance was identified together with the upland cotton genetic materials of GhUCE. It was found that GhUCE did improve the drought resistance of multiple crops ( Figure 7 In contrast, the Ghuce mutant was extremely sensitive to drought stress ( Figure 7 ). We believe that the GhUCE-SNPA allele has important breeding value, and future research can balance the contradiction between early maturity and high fiber yield or fiber quality in cotton by aggregating with multiple excellent loci.

Claims

1. A GhUCE gene for regulating plant development and drought resistance, characterized in that: Its nucleotide sequence is shown in SEQ ID No.

1.

2. An expression cassette, characterized in that: Comprising the gene according to claim 1.

3. A recombinant expression vector, characterized in that: It is a plant expression vector, which is obtained by connecting the gene fragment described in claim 1 to the vector Super-1300 or Cas9 to obtain the recombinant plasmids Super-1300-GhUCE and Cas9-GhUCE respectively.

4. A transgenic cell line, characterized in that: Comprising the gene according to claim 1.

5. The use of the gene according to claim 1, characterized in that: It is used in regulating crop growth period and drought resistance.

6. The use according to claim 5, characterized in that: The GhUCE gene is introduced into target crops for overexpression to obtain early-maturing and drought-resistant transgenic plants, wherein early maturation is specifically manifested in early flowering and early heading.

7. The use according to claim 6, characterized in that: The plants are sea island cotton, Asian cotton, upland cotton, tobacco, Arabidopsis thaliana, rice, wheat and the like.

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