Development Of Transgenic HaTLP Insect- Resistant Cotton

Cotton (Gossypium hirsutum L.) is one of the most important economic crops in the world. As we all know, cotton bollworm(Helicoverpa armigera) is one of the major pests that cause substantial losses in the yield of cotton. The application of transgene technique is an effective way to control pests. In recent years, the genetic engineering of cotton for insect resistance has developed rapidly. A series of insect-resistant transgenic plants were produced through genetic engineering, including Bt cottons. It is obvious that the development of Bt toxin-containing cotton has greatly decreased the yield loss of cotton production which caused by cotton bollworm. At the same time, it has brought remarkable economic, social and ecological effects. However, with the plantation of Bt plants, it is more and more serious that insects showed an increasing tolerance to Bt transgenic plants. Therefore, it is necessary for cotton insect-resistant breeding to find new effective insect-resistant genes, and to develop a safer method of insect resistant.Trypsin-like protease performs an important role in the protein digestion of an insect midgut, which is a member of the serine proteinase superfamily. It is considered a kind of endopeptidases.The cleavage occurs at the carboxyl side of the amino acids lysine and arginine within the polypeptide chain,but it may not happen when either is bound to a C-terminal proline.At the same time, it is also involved in the activation of other enzymes. The decrease or absence of trypsin-like protease could bring serious disruption to the growth and development of insects, and even cause the death of insects. In order to study the insect-resistant effect of HaTLP gene silencing on larval development, here we adopt a strategy based on RNA interference technology which will silence HaTLP gene of Helicoverpa armigera to develop insect-resistant cotton.The RNA interference vector (pCAMBIA2300::HaTLPRNAi) which contains HaTLP gene was transferred into cotton genome via the Agrobacterium-mediated method with WO as the receptor, and 89 clones were obtained by plant tissue culture technique.54 positive clones were selected from all clones by PCR analysis and the positive rate was 60.67%.45 positive To generation transformed lines were got after grafting. After the positive plants selfing,17 clones To generation seeds were harvested. One clone have got 34 T1 generation plants, and 20 of them were considered as positive plants based on the positive results of both PCR assay and kanamycin resistance identification.To study insect resistance of transgenic cotton expressing dsRNA of HaTLP, we fed cotton bollworm with some transgenic plants leaves. Bioassay for insect resistance with the To generation transgenic cotton plants showed that transgenic cotton plants had increased resistance to cotton bollworm. The leaf damage of transgenic plants was lighter than the control group. The development of cotton bollworm which were fed by transgenic cotton were delayed and they were distinctly smaller than the control group. The average weight of cotton bollworm which were fed by transgenic cotton were significantly lower than the control group, and all of 3 positive lines had a very significant difference (P<0.01) compared to the control. The new type of transgenic cotton we have got will become a new material for cotton insect-resistant breeding which have magnificent perspective.