阅读说明：本技术 棉花GhGOLS2基因在控制棉花种子萌发中的应用 (Application of cotton GhGOLS2 gene in controlling cotton seed germination ) 是由 师恭曜 田保明 位芳 郝梦媛 谢正清 田昭然 曹刚强 陈薇薇 高天奇 于 2021-06-22 设计创作，主要内容包括：本发明公开了一种棉花GhGOLS2基因在控制棉花种子萌发中的应用,棉花GhGOLS2基因在控制棉花种子萌发中的应用,棉花GhGOLS2基因作为Si-VIGS沉默系统阳性对照的应用。本发明通过使用携带TRV-GhGOLS2载体的两种侵染液分别浸染棉花种子,结果证明两种浸染液都可以有效沉默棉花GhGOLS2基因,并降低棉花种子的萌动率,病毒匀浆侵染液的沉默效果好于农杆菌侵染液的沉默效果,证明棉花GhGOLS2基因在控制棉花种子萌发中的应用。本发明证明GhGOLS2基因可以作为Si-VIGS种子萌发早期基因沉默研究,方便研究人员应用Si-VIGS方法研究基因功能。(The invention discloses application of a cotton GhGOLS2 gene in controlling cotton seed germination, application of a cotton GhGOLS2 gene in controlling cotton seed germination, and application of a cotton GhGOLS2 gene as a Si-VIGS silencing system positive control. According to the invention, the cotton seeds are respectively impregnated by two impregnation liquids carrying TRV-GhGOLS2 vectors, and the results prove that the two impregnation liquids can effectively silence the GhGOLS2 gene of the cotton and reduce the germination rate of the cotton seeds, the silencing effect of the virus homogenate impregnation liquid is better than that of the agrobacterium infection liquid, and the application of the GhGOLS2 gene of the cotton in controlling the germination of the cotton seeds is proved. The invention proves that the GhGOLS2 gene can be used for early gene silencing research of Si-VIGS seed germination, and is convenient for researchers to research gene functions by using an Si-VIGS method.)

1. The application of a cotton GhGOLS2 gene in controlling cotton seed germination is characterized in that: application of cotton GhGOLS2 gene in controlling cotton seed germination.

2. The use of the cotton GhGOLS2 gene according to claim 1 for controlling cotton seed germination, wherein the cotton GhGOLS2 gene comprises: the cotton GhGOLS2 gene is used as a positive control of a Si-VIGS silencing system.

Technical Field

The invention belongs to the technical field of cotton breeding, and particularly relates to application of a cotton GhGOLS2 gene in controlling cotton seed germination.

Background

Sugars in seeds are thought to be involved in the regulation and influence of the seed development process, among which Raffinose Family Oligosaccharides (RFOs) accumulate during seed maturation in most plants, and galactitol SYNTHASE (GOLS), Raffinose SYNTHASE (RS) and STACHYOSE SYNTHASE (STSs) are the major enzymes of plant RFO biosynthesis. Researchers found that contents of galactitol and raffinose are generally increased and tolerance of plants to abiotic stress is enhanced by expressing various GOLS genes in transgenic Arabidopsis or tobacco plants, so that the GOLS genes are widely researched. In 2016, Vidigal et al, after mutating the GOLS2 gene in cucumber, found that the reduction in galactitol content was consistent with a reduction in seed longevity, and therefore they considered GOLS2 as a suitable biomarker for seed longevity.

The patent discloses a method for silencing early genes of cotton, namely ' Si-VIGS ', with the patent number CN201910414489 ', and constructs a large number of pTRV2 transient silencing vectors through an ESTs library, cotton seeds are treated by using a Si-VIGS method, the phenotype of inhibiting the germination of the cotton seeds is found by observing the phenotypes of the cotton seeds and seedlings, then the gene sequencing is carried out on the pTRV2 transient silencing vectors, the sequencing result is subjected to BLAST search in a whole genome sequence of upland cotton, the gene fragment in the transient silencing vector is found to be matched with the CDS sequence information of the GhGOLS2 gene, and the primary judgment that the germination of the cotton seeds is inhibited after the GhGOLS2 gene is silenced is carried out. Si-VIGS is a simple, rapid and high-throughput virus-mediated gene silencing method, and can obtain silenced plants in the current generation. Si-VIGS advances the infection period of VIGS, and can research the genes related to the early growth and development of seeds and seedlings and underground parts. The Si-VIGS method shortens the time for inducing a gene silencing mechanism, thereby shortening the experimental period. In the commonly used method of VIGS, researchers have chosen the ghcala gene as a positive control, because once the ghcala gene is silenced, a photobleaching phenotype appears on cotton leaves after 7 days, which is very stable, so the ghcala gene is often used as a positive control in the traditional VIGS method, whereas the Si-VIGS method advances the action time of VIGS, at which point the cotton leaves are not opened, and the ghcala gene, which can whiten leaves after silencing, cannot be used as a positive control in the Si-VIGS method. Therefore, the Si-VIGS method needs to find a suitable positive control urgently. After the GhGOLS2 gene is silenced, the seed germination rate is reduced, the phenotype is relatively intuitive and is convenient for researchers to recognize, and the result of silencing the GhGOLS2 gene by using the Si-VIGS method is relatively uniform and stable.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art and provide an application of a cotton GhGOLS2 gene in controlling cotton seed germination.

The technical scheme for solving the technical problems is as follows: application of cotton GhGOLS2 gene in controlling cotton seed germination.

The cotton GhGOLS2 gene is used as the positive control of the Si-VIGS silencing system.

The invention has the following beneficial effects:

(1) according to the invention, the cotton seeds are respectively impregnated by two impregnation liquids carrying TRV-GhGOLS2 vectors, and the results prove that the two impregnation liquids can effectively silence the GhGOLS2 gene of the cotton and reduce the germination rate of the cotton seeds, the silencing effect of the virus homogenate impregnation liquid is better than that of the agrobacterium infection liquid, and the application of the GhGOLS2 gene of the cotton in controlling the germination of the cotton seeds is proved.

(2) The invention proves that the GhGOLS2 gene can be used for early gene silencing research of Si-VIGS seed germination, and is convenient for researchers to research gene functions by using an Si-VIGS method.

Drawings

FIG. 1 shows the result of PCR amplification on cotton GhGOLS 2.

FIG. 2 shows the results of the restriction enzyme of pTRV2-GhGOLS2 plasmid.

FIG. 3 is a representation of the phenotype of cotton plants after the silencing of the GhGOLS2 gene with a TRV-GFP vector control under Agrobacterium infection.

FIG. 4 shows the phenotype of cotton plants after the silencing of the GhGOLS2 gene with a TRV-GFP control under the treatment of virus homogenate infection.

FIG. 5 shows the germination rates of cotton seeds treated with Agrobacterium-infected fluid and virus homogenate-infected fluid for TRV-GFP control and GhGOLS2 gene silencing.

FIG. 6 shows the expression level of GhGOLS2 gene in cotton treated with both TRV-GFP control and GhGOLS2 gene silenced under the treatment of Agrobacterium infection and virus homogenate infection.

FIG. 7 is the GhGOLS2 gene expression analysis after Si-VIGS treatment of cotton seeds.

FIG. 8 shows the phenotype of TRV-GFP control and cotton plants after silencing GhGOLS2 gene after Si-VIGS treatment of seeds of three different varieties of cotton, Z79, ZG5 and Z9807.

FIG. 9 shows the expression levels of GhGOLS2 gene in TRV-GFP control and GhGOLS2 gene-silenced cotton after Si-VIGS treatment of seeds of three different varieties of cotton, Z79, ZG5 and Z9807.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The preparation method of the cotton GhGOLS2 gene of the embodiment is as follows:

CDS sequence design full-length primer of cotton GhGOLS2 gene:

GhGOLS2FL-F:5'-ATGGAAGGTGTTGCCATCGCCAAG-3'

GhGOLS2FL-R:5'-AGCAGCAGAGGGGGCGTTAATATA-3'

performing gene amplification by using dNTP, DNA Polymerase and DNA Polymerase Buffer of TAKARA company, and amplifying the total length of 993 bp; the DNA gel recovery kit from AXYGEN was used for purification and recovery, and the full-length gene was ligated into Ti vector for sequencing.

The construction method of pTRV2-GhGOLS2 transient silencing vector of the embodiment is as follows:

designing a specific primer of a gene silencing fragment according to CDS sequence information of the cotton GhGOLS2 gene, wherein the designed primer sequence is as follows:

GhGOLS2-F：5＇-GGAATTCGACCCAGTTTGCC-3＇

GhGOLS2-R：5＇-GGGTACCACCAAGTTGTAAATTG-3＇，

and adding restriction endonucleases KpnI and EcoRI at two ends of the primer respectively. The silent fragment is positioned in the middle region of the GhGOLS2 open reading frame, and the length of the amplified fragment is 441 bp. PCR amplification is carried out by taking root cDNA of upland cotton Z79 as a template, a target band of 441bp is obtained as shown in figure 1, the target band is connected to a pTRV2 vector, and the correctness of the recombinant plasmid pTRV2-GhGOLS2 is verified by enzyme digestion, wherein the vector fragment is 9663bp, and the target fragment is 441bp as shown in figure 2. Sequencing the recombinant plasmid which is verified to be correct by enzyme digestion, and transforming the pTRV2-GhGOLS2 transient silencing vector with the correct sequence alignment into the GV3101 agrobacterium cells.

Example 1

Experiment I, the inventor verifies the application of the GOLS2 gene in controlling the germination of cotton seeds through the following experiments.

Two staining solutions were used for this experiment: agrobacterium infection (aagrobacter), Virus homogenate infection (Virus sap);

subject: TRV-GFP control cotton plants are used as a control group, and cotton plants with GhGOLS2 gene silenced are used as an experimental group;

the specific steps of treating cotton seeds with the Si-VIGS method are disclosed in the patent number CN201910414489, entitled "A Cotton early Gene silencing method Si-VIGS".

As shown in the figures 3-4, the cotton seeds in the open white land are infected by the Si-VIGS method, the infected cotton seeds are transferred to wet vermiculite for culturing for 5d after being infected by the infection solution for 1d, and the seedlings are subjected to phenotype observation after being cultured for 5d, and experimental results show that the germination of the cotton seeds with the GhGOLS2 gene silenced is remarkably inhibited after the cotton seeds are treated by the two infection solutions by the Si-VIGS method.

As shown in fig. 5, the germination rates of the cotton seeds of the control group and the experimental group were counted, and the statistical results showed that: when the agrobacterium is used as the infection solution, the germination rate of the cotton seeds of the control group is 77%, and the germination rate of the cotton seeds of the experimental group is reduced to 18%; when the virus homogenate is used as the infection solution, the germination rate of the cotton seeds of the control group is 79 percent, and the germination rate of the cotton seeds of the experimental group is 13 percent.

As shown in fig. 6, to confirm that the GhGOLS2 gene was indeed silenced, the expression level of the GhGOLS2 gene in cotton seeds of the control group and the experimental group was further examined. The results of fluorescent quantitative PCR show that the two staining solutions can cause the silencing of GhGOLS2 gene in cotton seeds by a Si-VIGS method, thereby causing the phenotype of reduced germination rate of the cotton seeds. When the virus homogenate is used as an infection liquid, the reduction of GhGOLS2 gene expression level in cotton seedlings (seeds) is most remarkable.

In conclusion, the two infection solutions can effectively silence the GhGOLS2 gene of cotton and reduce the germination rate of cotton seeds, and the silencing effect of the virus homogenate as the infection solution is better than that of agrobacterium as the infection solution.

In order to further research the expression level of the GhGOLS2 gene in cotton seeds and determine that the Si-VIGS method can realize gene silencing in the early stage, the experiment carries out real-time fluorescence quantitative PCR on the cotton seeds in different growth stages.

Subject: upland cotton seeds with basically the same state are selected and divided into three groups: respectively, wild WT group, contrast carrying TRV-GFP infection solution as contrast group, and infection solution carrying TRV-GhGOLS2 vector as experimental group;

the cotton seeds are infected by using an agrobacterium infection solution and a virus homogenate infection solution in the experiment I through a Si-VIGS method for 0h, 1h, 3h, 6h, 9h, 12h and 24h respectively, and the cotton seeds cultured in wet vermiculite for 2d, 3d, 4d and 5d are taken as templates, RNA is extracted, the extracted RNA is reversely transcribed into cDNA, and qPCR is carried out by taking the cDNA as a template.

As shown in fig. 7, the expression level of GhGOLS2 gene in wild-type cotton seeds without any treatment increased significantly with the passage of time, the expression level of GhGOLS2 gene in wild-type cotton seeds at 3d of culture was about 40 times that at 0h of culture, and the expression level of GhGOLS2 gene in control group cotton seeds was substantially consistent with the change of wild-type; the expression level of the GhGOLS2 gene in cotton seeds which are silenced with the GhGOLS2 gene by using the Si-VIGS method is reduced after 6h infection, the expression level of the GhGOLS2 gene is reduced by more than 80% when the cotton seeds are cultured for 5d, and the expression level of the GhGOLS2 gene is reduced by 99% when virus homogenate is used as an infection liquid.

In conclusion, the Si-VIGS method realizes gene silencing after 6 hours of treatment, and the acting time of the Si-VIGS method is advanced.

Example 2

Experiment II, the inventor verifies the application of cotton GhGOLS2 gene as Si-VIGS silencing system positive control by the following experiment

Virus homogenate was used to invade the staining solution (Virus sap) separately in this experiment;

subject: selecting three cotton varieties with different genetic backgrounds of Z79, ZG5 and Z9807, taking a TRV-GFP carrier cotton plant as a control group and taking a cotton plant without GhGOLS2 gene as an experimental group;

the specific steps of treating cotton seeds with the Si-VIGS method are disclosed in the patent number CN201910414489, entitled "A Cotton early Gene silencing method Si-VIGS".

As shown in FIG. 8, the cotton seeds of the three varieties of the open upland cotton, namely Z79, ZG5 and Z9807 are respectively infected by an Si-VIGS method, the infected solution is infected for 1d and then transferred into wet vermiculite to be cultured for 5d, and after the infected solution is cultured for 5d, phenotype observation is carried out on seedlings, and experimental results show that after the cotton seeds are treated by the Si-VIGS method, the germination of the cotton seeds of different varieties of which the GhGOLS2 genes are silenced is remarkably inhibited.

As shown in fig. 9, to prove that the GhGOLS2 gene was indeed silenced in cotton seeds of different varieties, the expression level of the GhGOLS2 gene in cotton seeds of the control group and the experimental group was further examined. The results of the fluorescent quantitative PCR show that the Si-VIGS method can cause the silencing of the GhGOLS2 gene in the cotton seeds, thereby causing the phenotype of reduced germination rate of the cotton seeds.

In conclusion, the cotton GhGOLS2 gene can be silenced, the germination rate of cotton seeds can be stably reduced in various cotton varieties, and the stable and convenient observation phenotype enables the GhGOLS2 gene to be a proper positive control of the Si-VIGS method.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention.