| Verticillium dahliae Kleb is one of the most important diseases of cotton.In recent years,its occurrence and harm in China have been increasing year by year.However,cotton occupies a very important strategic position in the agricultural development of our country.Therefore,cultivating new varieties of cotton resistant to Verticillium wilt has great practical significance for ensuring the safe production of cotton in China.It is a very effective method to improve the resistance of cotton to Verticillium wilt in the upland and exogenous,and it is also an important way to ensure the sustainable development of cotton in the country.In the early stage of the laboratory,BS2 gene was isolated and cloned from Bactillus subtilis B111 against cotton deciduous Verticillium dahliae.The BS2 gene encodes a new antibacterial protein,which can inhibit the growth and reproduction of various fungi,especially it has the strongest inhibitory effect on Verticillium dahliae.Therefore,this study transferred the BS2 gene into tobacco and cotton,infected transgenic tobacco with Verticillium dahliae V991,and conducted a preliminary analysis of the resistance of BS2 gene to Verticillium wilt in tobacco.The main results were summarized as follows:1.The full length of BS2 gene is 1566 bp,encoding 521 amino acids.Among them,332 amino acids use non-plant preference codons,accounting for 63.72%.A total of 372 bases were optimized according to plant preference codons,involving 332 codons.The optimized protein amino acid sequence was identical with that before optimization.2.Bioinformatics analysis showed that the BS2 protein contains a glutamic acid active site(361-370 VTAHEMTHGV),which is classified as a neutral zinc metallopeptide enzyme.3.BS2 protein was located on cell membrane by subcellular localization.According to bioinformatics analysis and the pathogenic mechanism of Verticillium daliae,,BS2 protein is a secretory protein.4.The expression vector of pBI 121-BS2 was constructed and transformed into tobacco by leaf disc method.Nine BS2 transgenic lines were identified by PCR.On the PDA plate containing Verticillium dahliae V991,the oxford cup method was used to determine the resistance of the crude protein extract in transgenic tobacco,the crude extract of the transgenic strain produced obvious bacteriostasis circle,while the wild-type control did not produce bacteriostasis circle.5.Two transgenic lines with the highest expression level were selected for disease resistance analysis.The results showed that 2-4 true leaves of wild-type tobacco were completely yellow and the true leaves of transgenic tobacco were green after inoculation with Verticillium dahliae V991 for one week.The results of physiological and biochemical indices showed that the activities of SOD and CAT,as well as the contents of SA and ABA of the transgenic lines were significantly higher than those of wild-type tobacco after infection with Verticillium dahliae V991,indicating that the resistance of the transgenic lines to Verticillium dahliae was remarkable.It is higher than wild type.6.On the basis of transgenic tobacco experiment,overexpression vector pBI GR79-35S-BS2 and vascular bundle-specific promoter expression vector pBI GR79-vsp 1-BS2 were constructed.Agrobacterium-mediated transformation of cotton was carried out to improve cotton Verticillium wilt resistance. |
