Screening And Preliminary Analysis Of Gender-specific Genes Of Porphyra Yezoensis

Porphyra is one of the important economic algae in China and its output value is two of thirds of total algae value. Porphyra yezoensis which is bisexual develops with a linear way. The study of P.pururea is the only research of sex determination mechanism of Porphyra till now. To gain further understanding of sex determination mechanism and developing model of Porphyra, we made suppression subtractive hybridization experiments in some specialized genes which related to sex of Porphyra yezoensis are screened for the first time in this study.We extracted RNA of spermatangium cells and nutrition cells of Porphyra yezoensis as Tester and Driver respectively and got the cDNA using the method of SMART. The cDNA samples digested with Rsa I. Tester was divided into two groups. One group was ligated to adaptor 1 while the other to adaptor 2R. Two rounds of subtractive and PCR, we got the SSH library of gender-specific of Porphyra yezoensis. After transformation of the SSH library, we chose the positive clones in a way of blue/white spots screening and verified again using the method of colony PCR. Finally, we made a Dot blotting of the proper PCR products to check differences and sequenced.The results of sequencing which were analyzed on NCBI are as follows: After removing sequences of vector, fuzzy sequence and repeats from original sequences, we submitted 115 ESTs to NCBI(FG007280～FG007394). 5 clones are homologous to chloroplast genome sequences, 28 to nuclear genome sequences and 19 to cDNA or mRNA among 65 specific clones in spermatangium cell library while 11 clones are homologous to chloroplast genome sequences, 6 to nuclear genome sequences and 16 to cDNA or mRNA among 50 specific clones in nutrition cell library. There are 13 and 17 ESTs which have no homologous sequences in spermatangium cell library and nutrition cell library respectively and we presumed that they might represent new genes. In this study, we constructed SSH library of Porphyra yezoensis successfully and analysised the differences between spermatangium cells and nutrition cells. Since the researches of sex-determining mechanisms of Porphyra are few and the functions of majority genes are unknown, we only could speculate that proteins encoded by some genes maybe could influence or decide the way of sex development of Porphyra. There must be further study of the genes which play a critical role in sex determination. We provided a basis research of gender gene study in Porphyra yezoensis and created conditions to gain gender genes of Porphyra to culture monosexual Porphyra.