Analysis Of Genes Related To Self-compatibility In E.Sativa Mill By MRNA Differential Display Technique

Self-incompatibility is a widespread mechanism in flowering plants that prevents self-fertilization and promotes outcrossing. It has great influence on maintenance of germplasm purification and increasing in seed yield.The most effective method to resolve this problem are finding new gene resources of self-compatibility in Brassica and its relatives, analyzing their function and obtaining self-compatible plants, finally.To date, people could not find the nature of self-compatibility from the aspects of self-compatible genes. mRNA differential display technique is an efficient technique, its application in studying on self-compatibility can isolate differential expression genes from specific tissue,so as to understand the molecular mechanism of self-compatibility and apply them in breeding programmes. Leaves and stigmas at 5-leaf stage, 10-leaf stage, before anthesis, on the day of anthesis, after anthesis from self-compatible E.sativa Mill were used, accordingly, the related tissues of self-incompatible lines as control. With 3 anchoring primers and 10 random primers, specific cDNA fragments which may be closely related to self-compatibility were isolated with mRNA differential display technique. The results are as the following:1.Several hybridized combinations were made between self-compatibility lines and self-incompatibility lines, to analyse the hereditary characteristics of self-compatibility in E.sativa Mill. The results showed that all the F1 plants were self-incompatible, and the F2, BC1F1 populations segregated in ratio.Through chi square test, the segregation ratios of self-compatibility to self-incompatibility in observed F2, BC1F1 populations were coincided with the ratio 1:3, 1:1, respectively. It was inferred therefore that the hereditary characteristics of self-compatibility in E.sativa Mill was controlled by one recessive nucleus gene.2.Total RNA extracted by improved guanidine thiocyanate method and treated with DNaseⅠwere found to be applicable to mRNA differential display technique. Its absorbance ratio OD260/OD280 was ranged from 1.9 to 2.1, which demonstrated that RNA samples were free of contamination of DNA, protein. Agarose electrophoresis showed that RNA samples thus-obtained were in good integrity. 3. An economical reaction system of purification of total RNA was put forward in this study. In order to remove chromosome DNA, two different systems were compared. The results indicated that both RNA samples purified could be used as templates for cDNA synthesis. One system without Ribonucleasei inhibitor was chosen, thus its cost was considerably declined.The purification systems was: 20μL total RNA; 2.5μL 10×DNaseⅠB uffer; 1μL DNaseⅠ(5U/μL); 1.25μL DEPC-H2O; 37℃30 min .4. An effective method of mRNA differential display for E.sativa Mill.was established in the investigation.The factors affected the mRNA differential display reaction system was set up.This system was 25μL total volume contained 17.2μL DEPC-ddH2O; 1.5μL 10×PCR Buffer (plus Mg2+); 2.0μL dNTPs (each 2.5mM); 1.0μL anchoring primer (10μM); 1.0μL random primer (5μM); 1.5μL cDNA; 0.8μL Taq DNA Polymerase(2.5U/μL).mRNA differential display steps: 1) 94℃4 min; 2) 30℃1 min 30s; 3)72℃1 min; 4) 94℃30s; 5) 42℃1 min 30s; 6)72℃1 min; from 4) to 6) 35 cycles; 7)72℃10 min.5.The results of mRNA differential display on leaves at 5-leaf stage, 10-leaf stage, before anthesis, on the day of anthesis and after anthesis found that amplified cDNA fragments using every pairs of primers were the same among the self-compatible lines and self-incompatible lines at the same growth period, which showed tissue specific expression patterns of self-compatible genes and that they were not expressed within all the tissues, especially which were not correlated with the state of development of the leaves.6.With 30 pairs of primers, mRNA differential display on stigmas from self-compatible lines and self-incompatible lines before anthesis were performed. 142 cDNA fragments were obtained, of which 90 ones could be seen in self-compatible lines, 52 ones in self-incompatible lines.The great majority of bands amplified were 200～1500bp. Self-compatible lines had 10 differentially expressed bands of which only 2 bands exhibited stably with different pairs of primers,the one was less than 100bp,the other was 750～1000bp; in addition, 6 cDNA fragments appeared in self-incompatible lines, only one 750bp band generated repeatedly.All 133 cDNA fragments were obtained in mRNA differential display products of sigmas on the day of anthesis.Of which 87 ones exhibited in self-compatible lines and 46 ones in self-incompatible lines. Besides two specific bands which existed before anthesis, another 300bp new differential band was found unique to self-compatible lines. At the same time, one 500～600bp band was identified in self-incompatible lines.The results of mRNA differential display on stigmas after anthesis were identical to those observed on stigmas on the day of anthesis. The study also found that anchoring primer H-T11C was more efficient than others in the identification of differentially expressed bands.Sum up, three repetitive and steady differential cDNA fragments were presented in stigmas of self-compatibility,which might be closely related to the self-compatible genes in E.sativa Mill.