Study On Cloning And Genetic Transformation System Of Cotton Fiber Specific Promoter

Some studies have found that many genes are expressed in cotton fiber and strictly control the growth and development of cotton fiber.As pigment molecules are deposited in the cotton fiber cavity during the development of the cotton fiber,the color of the cotton fiber appears.The color of cotton fiber can be changed by transforming cotton into a plant expression vector that drives pigment gene by cotton fiber specific promoter.Therefore,this study cloned the Gh GRPL1 promoter of cotton fiber development specific gene from cotton,and verified the activity of the promoter.Therefore,transformation research was carried out.An efficient genetic transformation system was established,which laid a foundation for genetic transformation of vectors.The experimental results are as follows:1. Cloning and Analysis of Cotton Fiber Specific Gene Gh GRPL1 PromoterIn order to obtain the promoter of cotton fiber-specific gene Gh GRPL1,this study extracted the genomic DNA of the high-yield,insect-resistant upland cotton Jingzhou main variety"Chuang 075"and cloned the promoter by PCR technology,and The promoter sequence was 2078 base pairs long.Sequence analysis of the promoter of the Gh GRPL1 gene.This promoter contains multiple promoter core elements TATA-box and CAAT-box,multiple light-responsive elements?ATC-motif,Box4 and G-Box?,and three abscisic acid responses Element?ABRE?,1 salicylic acid response element?TCA-element?,1 auxin response element?TGA-element?,3 cis elements?ARE?necessary for anaerobic induction and a few DRE cores,MYB and other components with unknown functions.Constructed a fusion vector that deleted the 5'end of the promoter to drive the expression of the GUS gene.This vector infects cotton leaves through Agrobacterium rhizogenes ATCC15834,and obtain hairy roots of transgenic cotton,The GUS staining experiment results showed that the transgenic hairy roots were all stained blue.This indicates that the promoter of Gh GRPL1 is active.2. Preliminary study on cotton regeneration system in vitroIn order to establish a cotton regeneration system by cell pathway,11 cotton varieties were screened,and the callus induced by Jingzhou main variety"Chuang 075"was found to be the best.This callus was induced on MSB medium supplemented with0.1 mg/L 2,4-D+0.1 mg/L KT,and the recovery rate reached 100%.Beige granular embryogenic callus is formed on the differentiation medium.Add 0.2 mg/L NAA or 0.3mg/L IBA to the differentiation medium,the embryogenic callus can differentiate into a more complete root system.In order to establish a cotton regeneration system by organ pathway,adding 0.5 mg/L 6-BA to the differentiation basic medium can induce7-day-old cotton"Chuang 075"shoot tips to produce buds,and the bud induction rate is as high as 96.67%.In addition,the addition of about 1.0 mg/L of NAA or 0.8 mg/L of IBA to 1/2MS medium can induce root generation,and the root induction rate is between 30-40%.3. Preliminary establishment of cotton genetic transformation systemIn order to establish an efficient and stable cotton in vitro genetic transformation system,the hypocotyl of Jingzhou main cotton variety"Chuang 075"was taken as the research object,and the EHA105 strain containing the p CAMBIA1300G vector was used as the transformed strain.The effect of concentration,Agrobacterium infection time and co-cultivation time on the induction of callus was selected,and the optimal culture conditions for the occurrence of resistant callus were screened out.The genetic transformation system of cotton"Chuang 075"hypocotyls is:adding 100?mol/L AS in the infection solution,the OD₆₀₀ of the bacterial solution is 0.6,the infection time is 15min,and the co-culture is 48 h.In addition,the sensitivity test of the screening agent was carried out.The results showed that adding 80 mg/L Kana or 15 mg/L hyg or 8mg/L PPT to the selection medium could inhibit the occurrence of callus.4. Obtaining transgenic cotton cellsIn order to obtain cotton cell clusters containing foreign aid genes,the EHA105strain containing the p CAMBIA1300G vector was used as a transformed strain,and the cotton hypocotyls were infected under the optimal genetic transformation conditions.26transgenic calli from 60 explants were selected by hygromycin and the conversion rate was 43.33%.The EHA105 strain containing the recombinant vectors of p CAMBIA1300-p GRPL1-At3GT and p CAMBIA1301-p GRPL1-Gh DFR as transformation strains was used to infect the cotton hypocotyl under the most suitable genetic transformation conditions.5 groups of callus obtained from 60 infected hypocotyls were screened by hygromycin and glufosinate.Detected by PCR technology,there are 3 cell clusters that all contain marker gene bands.Therefore,3 groups of transgenic cells were obtained with a total transformation rate of 5%.In summary,this study successfully isolated the promoter of the cotton fiber-specific gene Gh GRPL1,and the construction of GUS fusion expression vector verified the activity of the promoter.Initially established the regeneration system of upland cotton"Chuang 075"varieties through tissue culture technology,At the same time,a more complete genetic transformation system of"Chuang 075"cotton varieties was established,The results of these studies laid the foundation for the genetic transformation of"Chuang 075"cotton varieties.