Complementary CDNA Library Construction, Sequencing And EST Analysis Of Cotton Fiber/Ovule In Gossypium Barbadense L.

As a major source of industry fiber,cotton is an important economic crop playing an important role in the global economy,cDNA library construction and screening is an efficient for gene cloning,which is a basic tool for gene finding and function research currently.Target genes can be fished from cDNA library,and directly used biotechnologically.In this research,using the mRNA from the ovules(fiber attached) of -3, 0,3,and 6DPA of Hai7124,a mixed cDNA library was constructed and 11,970 clones were arbitrarily picked out for sequencing based on T7 primer,based upon the obtained ESTs sequence data,the function of the genes expressed during the cotton fiber initiation and early elongation were predicted in silica,EST-SSR markers were developed,localized and mapped.1.Construction and EST sequencing of Hai 7124 cDNA libraryUtilizing the SMART technique of the Clontech company,we constructed the cDNA library of sea island cotton Gossypium Barbadense cv.Hai 7124 of the ovules(fiber attached ) of -3,0,3,6DPA.We randomly isolated 11,970 clones for 5' end sequencing using the primer T7 of the vector,and obtained 9,034 ESTs sequence,8,783 of those with a length of more than 500bp as a result of chromas software analysis,furthermore,we obtained 3,505 Unigenes jointed with Cap3 program.The sequence similarity between the 3,505 Unigenes and the 32,190 ESTs of TM-1 downloaded from NCBI database were analyzed by using BLASTn program with the E-value=1e-100,1,110(31.67%) Unigenes can find matches in TM-1 database but 2,395 (68.33%) has no match,when this compare was made with the whole 281,233 ESTs of cotton,3,173(90.53%) Unigenes can be matched while only 332(9.47%) has no matches.2.Function and metabolism analysis of the UnigenesUsing the BLAST2GO online software,the function and metabolism pathway of the 3505 Unigenes were analyzed.The results reveal that 1574(44.9%) Unigenes involved in metabolic process,including carbohydrate metabolism(314,19.9%) and amino acid metabolism(246,15.6%),according to the function analysis,the Unigenes fall into three categories:(1) cellular component,(2) molecular function,and(3) biological process,the top two of each category are cell(59%) and organelle(26%),binding(41%) and catalytic activity(37%),and cellular process(30%) and metabolic process(26%) respectively.3.EST-SSR marker development and mappingThe 3,505 Unigenes was applied for EST-SSR detection using the AutoSSR software, and 191(11.38%) Unigenes were found to contain 208 SSR,52 pairs of primers were consequently designed from the 191 ESTs utilizing the online software of primer 3.TM-1 and Hai7124 genome DNA were subjected for PCR analysis using the 52 pairs of the designed primers,and 23 pairs of primers produced polymorphisms,the BC1 population of TM-1 and Hai7124 was further,the results reveal that 18 pairs of primers produce 19 Mendelism polymorphism alleles which mapped to 10 chromosomes,5 pairs of primers exhibit distorted segregation.