Application of GhSPL7b-A1 gene in salt tolerance of cotton

Abstract

The application belongs to the field of genetic engineering, and particularly relates to application of a GhSPL7b-A1 gene in salt tolerance of cotton, comprising construction of a GhSPL7b-A1 overexpression vector, genetic transformation of cotton hypocotyls by means of agrobacterium, obtaining of transgenic overexpression pure line plants, and disclosure of a gene sequence of the GhSPL7b-A1. The application of the GhSPL7b-A1 gene will promote genetic improvement of cotton planting in saline-alkali soil and improvement of high-quality fibers, provide theoretical innovation and technical progress for cultivation of new cotton varieties with further improved salt tolerance, and provide excellent resource support for improvement of utilization efficiency of saline-alkali soil.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to the application of the GhSPL7b-A1 gene in the salt tolerance of cotton. Background Technology

[0002] There is an urgent need to conduct systematic research on salt and alkali tolerance phenotypes, physiology, and genetic regulation, and to discover key genes that synergistically regulate salt and alkali tolerance and promote fiber quality and yield. This is of great significance for breeding high-yield and high-quality salt and alkali tolerant new cotton varieties. Summary of the Invention

[0003] In order to solve the problems existing in the prior art, the present invention provides an application of the GhSPL7b-A1 gene in the salt tolerance of cotton, which can improve the salt tolerance of the plant and increase the fiber strength of cotton.

[0004] The specific technical solution adopted in this invention is as follows:

[0005] An application of the GhSPL7b-A1 gene in the salt tolerance of cotton was described. A GhSPL7b-A1 overexpression vector was constructed, and cotton hypocotyl genetic transformation was mediated by Agrobacterium to obtain transgenic pure lines of overexpressing GhSPL7b-A1. The gene sequence of GhSPL7b-A1 is as follows:

[0006] The amino acid sequence encoded by GhSPL7b-A1 is MNKDFIAEGLDNDMQEEEEEGVGGDHGFPDDEKKKKGYGRRGAAGGGGGV SPPACQVEKCGLDLSDAKRYHRRHKVCEIHAKAPFVVVAGLRQRFCQQCS RFHELPEFDEAKRSCRRRLAGHNERRRKSSAESSSAAESSSRRGMMISAQ

[0007] LKESHYLADDQRARVNPMAIHGSSSFKRSQIR.

[0008] A gene vector based on GhSPL7b-A1 was constructed and transformed into a plant host cell to obtain a transgenic plant; the plant was cotton.

[0009] The transgenic pure-line plants were self-pollinated to screen for stable germplasm materials as germplasm for new varieties of salt-tolerant cotton.

[0010] The beneficial effects of this invention are:

[0011] The application of the GhSPL7b-A1 gene involved in this invention will promote the genetic improvement of cotton planting and high-quality fiber synergistic improvement in saline-alkali land, provide theoretical innovation and technological progress for promoting the breeding of new cotton varieties with further improved salt tolerance, and provide excellent resource support for improving the utilization efficiency of saline-alkali land. Attached Figure Description

[0012] Figure 1 For subcellular localization and expression analysis

[0013] Figure 1 A. Subcellular localization (from left to right: bright field, fluorescence channel, and overlay image); Figure 1 B. Analysis of expression in different tissues and at different time points after NaCl treatment; Figure 1 C. Expression induced by different concentrations of NaCl; Figure 1 D. Expression induced by different concentrations of seawater;

[0014] Figure 2 Phenotypic and expression levels of VIGS-silenced plants (A), a. V2 empty vector; b. Silent plants; scale bar, 7 cm; and H2O2 and SOD detection (B).

[0015] Figure 3 Cotton transformed with GhSPL7b-A1 overexpression vector and Southern spectroscopy detection

[0016] Figure 3 A. Hypocotyl; Figure 3 B. Embryonic callus; Figure 3 C. Germinating embryo; Figure 3 D. Regenerated plants; Figure 3 EMDNA marker, P.GhSPL7b-A1 positive plasmid, others are single-strain numbers;

[0017] Figure 4 Gene expression levels and phenotypic detection of GhSPL7b-A1 overexpression lines

[0018] Figure 4 A. Expression level in overexpression lines; Figure 4 B. Fiber specific strength; Figure 4 C. Length of the upper half of the fiber; Figure 4 D. Root length before and after salt stress; Figure 4 E. Fresh weight of sprouts before and after salt stress; Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments:

[0020] This invention provides an application of the GhSPL7b-A1 gene in the salt tolerance of cotton, achieved by constructing cotton plants overexpressing GhSPL7b-A1. The gene sequence of GhSPL7b-A1 is as follows:

[0021] The amino acid sequence encoded by GhSPL7b-A1 is MNKDFIAEGLDNDMQEEEEEGVGGDHGFPDDEKKKKGYGRRGAAGGGGGV SPPACQVEKCGLDLSDAKRYHRRHKVCEIHAKAPFVVVAGLRQRFCQQCS RFHELPEFDEAKRSCRRRLAGHNERRRKSSAESSSAAESSSRRGMMISAQ

[0022] LKESHYLADDQRARVNPMAIHGSSSFKRSQIR

[0023] This application obtained 226 SPL family genes by performing whole-genome alignment of SPL family transcription factors, including 60 from upland cotton, 54 from sea island cotton 3-79, 53 from sea island cotton Xinhai 21, 29 from Asian cotton, and 30 from Gossypium raemondii. Bioinformatics analysis was performed, and the GhSPL7b-A1 gene was located on chromosome 10.

[0024] In previous studies, the inventors' research group discovered, through combined analysis of expression profiles of 8 DPA and 20 DPA in high-quality upland cotton Ji 228 fibers and small RNA sequencing, that the candidate gene GhSPL7b-A1 (formerly named GhSBP3, KJ569110) was predominantly expressed at 20 DPA and 25 DPA during fiber development, showing a trend of first increasing and then decreasing.

[0025] This application determined the nuclear location and salt-induced expression pattern of a gene through subcellular localization and salt-induced expression analysis. A gene vector based on GhSPL7b-A1 was constructed, specifically a GFP fusion vector, which was then transformed into tobacco cells. The tobacco cells were observed under an FV10-ASW laser confocal microscope (OLYMPUS). The results showed that the gene was located in the nucleus (…). Figure 1 A), consistent with expectations. Tissue-specific expression induction analysis of this gene after NaCl stress was performed ( ). Figure 1 B), except for no significant increase in gene expression in the stem, the gene expression in the leaves and roots showed a significant increasing trend. The expression level in roots peaked after 3 hours of stress, while in leaves it peaked after 6 hours. Analysis of NaCl-induced expression at different concentrations showed that, when sampled from the whole plant after 6 days of growth in NaCl hydroponics, the gene expression level was significantly enhanced at 100 mM-200 mM. Figure 1 C). Induction with different concentrations of artificial seawater also showed that the expression level of this gene significantly increased with increasing seawater concentration. Figure 1 D).

[0026] Furthermore, this application has determined that the GhSPL7b-A1 gene positively regulates salt tolerance through genetic transformation and silencing of the GhSPL7b-A1 gene.

[0027] Specifically, the VIGS vector for GhSPL7b-A1 was constructed, and GhSPL7b-A1 was silenced in upland cotton, resulting in a significant decrease in the expression level of this gene. Figure 2 A), after 24 h of 150 mM NaCl stress, compared with the control, plants with silenced GhSPL7b-A1 showed increased salt sensitivity and decreased salt tolerance. Silent plants also showed significantly increased H2O2 and significantly decreased SOD activity after salt stress. Figure 2 B).

[0028] Overexpression vectors and gene editing vectors for GhSPL7b-A1 were constructed to obtain transgenic and edited plants, resulting in transgenic positive and single-copy plants. Figure 3 ).

[0029] Expression levels were detected in transgenic overexpression plants. Figure 4 A) and fiber quality testing showed that the improvement in fiber strength reached a highly significant level. Figure 4 B, C). Lines with high expression levels were selected, and germination experiments were conducted using 25% artificial seawater. The results showed that, compared with the control, the seeds overexpressing GhSPL7b-A1 were less affected by salt stress during germination, and the radicle elongation was significantly greater than that of the control. Figure 4 D) Fresh weight was significantly higher than the control ( Figure 4 E).

[0030] In summary, this gene was induced by NaCl and artificial seawater, and VIGS found that silencing this gene reduced the plant's salt tolerance. The transgenic overexpression cotton lines showed improvements in both fiber length and strength, with a significant increase in fiber strength. Compared to the control, the overexpression lines exhibited significantly stronger seed germination under salt stress.

Claims

1. Application of GhSPL7b-A1 gene in improving salt tolerance of cotton, characterized in that: A GhSPL7b-A1 gene overexpression vector is constructed, and a transgenic overexpression pure line plant is obtained through Agrobacterium-mediated cotton hypocotyl genetic transformation, wherein the nucleotide sequence of the GhSPL7b-A1 gene is shown as SEQ ID NO:

1.

2. Use according to claim 1, characterized in that: The amino acid sequence coded by the GhSPL7b-A1 gene is shown as SEQ ID NO:

2.

3. Use according to claim 1, characterized in that: The transgenic pure line plant is self-crossed to be homozygous, and a germplasm material with stable traits is screened as a new variety of high-salt-tolerance cotton germplasm.

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