Study On Cotton(Gossypium Hirsutum) Genetic Transformation And The Role Of The LIM Genes In Transgenic Cotton Progenies

Cotton is the most important fiber crop in the world.The production of cotton is closely related to the development of agriculture,handicraft,trade industry and other industries.The quality of cotton fiber determines the commercial production value of cotton fiber.Exploring the molecular regulation mechanism of cotton fiber cell elongation and secondary wall synthesis has important theoretical and practical value for improving cotton fiber quality.Five LIM proteins family genes have been identified in previous studies in our laboratory,we obtained transgenic cotton plants and their descendant lines of GhWLIM5 and GhXLIM6.In this paper,we discussed the function and regulatory mechanism of two LIM proteins in the fiber development of transgenic cotton descendant lines in depth.In addition,we also analyzed a MYB transcription factor gene that was predominantly expressed during the secondary wall development of cotton fiber cells,and constructed the vector,transformed into cotton to obtain the transgenic cotton line of the MYB gene.The main results are as follows:1?Inhibiting the expression of Gh WLIM5/GhXLIM6 hinders the elongation and development of cotton fiberIn order to research the function of GhWLIM5 and GhXLIM6 during cotton fiber development,we analyzed the genetic phenotypes such as fiber length changes of descendant lines of GhWLIM5 and GhXLIM6 RNAi transgenic cotton.We measured the length of mature cotton fibers from different RNAi cotton descendant lines,the statistical analysis showed that the length of mature cotton fiber of transgenic cotton was higher shorter than that of wild type with different degrees.Further,the length of the developing cotton fiber cell was measured by in vitro culture of the ovule,and it was found that the growth rate of the transgenic cotton fiber was significantly slower than that of the wild type during the rapid elongation of the fiber.These results suggest that GhWLIM5 and GhXLIM6 may play a positive regulatory role in the elongation phase of cotton fibers.2?GhWLIM5 and GhXLIM6 act as actin binding proteins to promote F-actin aggregation into bundles in cotton fibersIt is necessary for F-actin to maintain a certain degree of polymerization during cotton fiber development.Treatment of wild type cotton ovules with LatB(F-actin depolymerizing agent)would hinder cotton fiber development.Using Atto 488-Phalloidin to stain F-actin in cotton fibers in rapid elongation phase,the results showed that the actin filaments were thinner in the cotton fibers of GhWLIM5 and GhXLIM6 RNAi transgenic plants compared with wild-type cotton fibers.The quantitative analysis also showed that the F-actin polymerization degree was lower in these transgenic cotton fibers.These results indicate that both GhWLIM5 and GhXLIM6 can act as actin-binding proteins to promote the formation of F-actin bundles in cotton fiber cells,thereby affecting the elongation and development of cotton fibers.3?The capacity of GhWLIM5 binding to F-actin and promoting F-actin polymerization are different with GhXLIM6Previous studies have shown that different LIM proteins may have different abilities of binding to F-actin and promoting F-actin polymerization.This study we compared the ability of GhWLIM5 and GhXLIM6 proteins binding to F-actin by quantitative high-speed co-precipitation experiments firstly.The results showed that with the addition of the same concentration of F-actin,GhXLIM6 has a higher proportion in the precipitation,while the precipitation amount of GhWLIM5 is significantly lower than the former,which indicates that GhXLIM6 has a stronger binding capacity with F-actin.Subsequently,we used a low-speed coprecipitation experiment to compare and analysis the ability of GhWLIM5 and GhXLIM6 to promote F-actin polymerization.The results showed that the addition of 2?M of GhXLIM6 allowed 70%the low polymerization degree F-actin to be polymerized into F-actin bundles of a high polymerization degree,while GhWLIM5 required 4?M to achieve the same effect.This indicated that GhXLIM6 was stronger than GhWLIM5 during promoting the F-actin polymerization.4?The ability of LIM protein to promote F-actin polymerization is affected by pH valuePrevious studies have shown that the ability of some LIM proteins promoting the F-actin polymerization affected by pH value.Therefore,we used low-speed co-precipitation experiments to compare the ability of these two LIM proteins to promote F-actin polymerization under different pH conditions.The results showed that when the same concentration of GhWLIM5 was added,the proportion of F-actin in the precipitate increased with the decrease of pH value,indicating that GhWLIM5 promoted the polymerization of F-actin were affected by pH value,the lower the pH value,the stronger the ability of promoting polymerization.In contrast,experimental result demonstrates pH value changes did not affect GhXLIM6's ability to promote F-actin polymerization.5?Genetic transformation of cotton and identification of transgenic cotton plant of GhMYBLlWe isolated and identified a MYB transcription factor gene GhMYBL1 that expressed in cotton fiber during the secondary cells wall development predominantly.Then we constructed GhMYBL1 overexpression and RNAi vectors,obtained GhMYBL1 overexpression and RNAi transgenic cotton plants by agrobacterium-mediated cotton transformation,and got a total of 65 positive seedlings of 30 strains,transplanted to the field or greenhouse,and further grown to maturity subsequently.Analysis of the genetic phenotype of GhMYBL1 transgenic cotton lines is ongoing.6?Recombinant expression of GhMYBLl protein and polyclonal antibody preparationWe constructed the prokaryotic expression vector pET28a-GhMYBL1,induced the expression of GhMYBLl protein in E.coli,obtained polyclonal antibody of GhMYBLl by immunizing rabbits,and detected the specificity and titer of the antibody by Western Bloting,indicating that the antibody can be used for subsequent related experimental studies.