| Objective: Xinjiang is the only Sea Island cotton production area in our country. Sea island cotton has already become the main raw material of high-grade textile because of it’s higher quality fiber. But the problem caused by Fusarium Oxysporum f.vasinfectum of sea island cotton is becoming more and more serious and has hindered the development of sea island cotton in Xinjiang. Thus, selecting and breeding effective resistance Sea Island cotton varieties became urgent affairs, while modified genetical technology provides a perfect method to achieve this goal. In this study, a WRKY transcription factor gene was segregated from Gossypium hirsutum cultivar, and preliminary function research was set up for providing the basis theory for resistance to Fusarium wilt and achieving genetic resources for cotton genetic engineering breeding.Results: 1. A WRKY gene was cloned from Gossypium hirsutum cultivar "Zhongmiamsuo12". According to blasting in NCBI, the cloned gene sequence was similar as high as 99.24% to the sequence of GhWRKY106, so named the gene Gh WRKY106-1(Accession:KP202869.1). The length of the gene’s ORF was 921 bp, encoding 306 amino acids. The WRKY protein has a conservative WRKY domain in N-terminal and a CX7CX24 HC type zinc finger structure in the C-terminal, indicate that the WRKY gene belongs to the Ⅲ WRKY subfamily. The protein is an unstable hydrophilic protein which has 35.30 KD molecular weight and a theoretical isoelectric point of 7.10. 43.00% random extern and 32.57% alpha helix compose the main Secondary structure components of the protein.2. Real-time quantitative PCR analysis showed that GhWRKY106-1 can be induced by Fusarium. However, the expression of the gene is far higher in susceptible variety than in resistant variety. In hormone treatments, Gh WRKY106-1 could response to JA, SA and ET Signaling molecules, speculated that GhWRKY106-1 may participate in cotton Fusarium pathogen resistance through multiple signal transduction pathways.3. After constructing the over-expression cloning vector, the gene was transferred into tobacco by Agrobacterium-mediated transformation method-leaf disc method to study the basic functions of the gene. 31 transgenic tobacco plants were verified in T1 generation through Kanamycin screening and PCR identification methods. OE1 transgenic plants were used to test the expression of downstream PRs gene. The results showed that the expression of PRs gene were different in transgenic line.In the absence of Fusarium, the expression of NtPR1 a and NtPR5 were lower in OE1 than in WT tobacco, while the expression of NtNPR1 have no significant difference.When treated with Fusarium, the expression of PRs gene in transgenic line was much lower than in WT at the same period of the inoculation except NtPR1 a gene. In other words, Gh WRKY106-1 may act as a negative regulator which can repress down-regulated genes expression to take part in cotton Fusarium resistance.4. Using VIGS technology to silence GhWRKY106-1 gene, the expression of downstream PRs genes increased obviously in Zhongmiansuo12 slicenced plants compared with none slicenced plants; After Fusarium inoculation, the resistance to Fusarium increased greatly in slicenced cotton plants.5. In fields experiment, the GhWRKY106-1gene was transferred into sea island cotton variety Xinhai 14 through Agrobacterium-mediated transformation flower stigma invasion method. After eliminating with Kanamycin and PCR method screen, 4 transgenic cotton plants were identified. The genetically modified positive conversion rate was 1%.Conclusions: GhWRKY106-1 participated in the response to Fusarium wilt, JA SA, and ET signal molecules in cotton, and may be a WRKY transcription factors associated with resistance to Fusarium wilt pathogen. The level of Gh WRKY106-1 in transgenic tobacco and cotton can regulate the expression of PRs genes and manipulate the disease resistance in the plants. More particular functions remain to be further explored. |
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