Expression Characteristics And Functional Analysis Of GbNPR1Gene From Sea Island Cotton

Objective：Sea Island cotton is one of China’s important cultivars, Xinjiang as China’s majorcotton growing area, its favorable natural conditions provide a unique advantage for thedevelopment of Sea Island cotton. However, it is easy to be affected by fungi, bacteria and virusin the growth process, so it will suffer seriously blight, which will seriously affect the yield andquality of cotton. Therefore, we use modern genetic engineering techniques to dig resistance generesources, and to study its function in disease resistance in Sea Island cotton, which will layfoundation for blight-resistant Sea Island cotton varieties breeding.Method: In this study, with Sea Island cotton14,16and21in Xinjiang as the materials, we usedRT-PCR technology to tap and exploit blight-resistant-related gene GbNPR1, and carried onbioinformatic analysis of the gene; we use real-time PCR technology to analyze the geneexpression levels in Fusarium stress and salicylic acid and ethylene treatment; Constructed theover-expression plant vector, transfered the gene to Sea Island cotton by Agrobacterium-mediatedtransformation of shoot tips, for a preliminary study of its functions; We used Agrobacteriuminfecting stigma method and Agrobacterium buds injection method for Sea Island cotton genetictransformation in the field, to explore an effective genetic transformation method.Results:1.We clone the GbNPR1gene from Sea Island cotton, which has a1764bp full-length openreading frame, encodes587amino acids, its molecular weights65393.8, and its theoreticalisoelectric point is5.99, the protein-coding gene is a hydrophilic protein; it contains a BTB/POZdomain, three ankyrin repeats (ANK) domains and the representative NPR1_like_C domains ofNPR1protein family. Random coil and α helix in the secondary structure of the protein shares aportion of42.42%and49.40%, respectively.2. qPCR analysis showed that, after fusarium induction, GbNPR1gene expression in resistant andsusceptible varieties are significantly increased, the highest values are up to7.2times and5.4times before induction, but the expression in the resistant material H116H-1is generally higherthan in the susceptible varieties; GbNPR1genes in disease-resistant varieties significantly respondto salicylic acid and ethylene, but the gene in susceptible material doesn’t change significantly forethylene-induction. Speculating GbNPR1gene may play a certain role in blight-resistant in cotton.3. Through Stem tip genetic transformation, we obtain35transgenic cotton individuals, after PCRtesting,21individuals are positive strains (16ndividuals from Xinhai14,5individuals fromXinhai21), the positive rate is60%. qPCR detection discovers that, after Fusarium oxysporumtreatment, GbNPR1gene expressing quantity in positive transgenic plants is significantly higherthan non-transgenic plants.4. It shows that, Agrobacterium infecting stigma method and Agrobacterium buds injection method both can obtain positive plants after kanamycin and PCR testing, we obtain12and8positive plants, respectively, positive conversion rates are3.08%and2.00%by the two methods,correspondingly, both higher than the traditional pollen tube pathway.Conclusion: Studies have shown that genes involved in the Sea Island cotton GbNPR1responsedto Fusarium, salicylic acid and ethylene-induced reaction, it may play a role in Island cotton andFusarium interaction effects. But its exact function needs further study. In addition, Agrobacteriuminfecting stigma method and Agrobacterium buds injection method offer new ways to establish asimple, rapid and efficient genetic transformation method.