Expression And Cloning Of Differential Genes Associated With The Self-compatibility Of Eruca Sativa Mill

Eruca sative Mill is an Sporophyte self-incompatibility plant, With the excellent resistance ability.It is an important gene resources of cruciferous crops breeding. Self compatibility can make the plants to achieve self in the natural condition, in favor of the beneficial to carry the gene retention and accumulation, so that the plants themselves enhance disease resistance, improve the yield; and also conducive to the breeding of excellent inbred lines, to ensure stable germplasm pure, increase varieties, prolong the service life of the seed. Therefore, Eruca sative Mill selfed studies have found that the affinity gene resource genetic mechanism and cloning of self incompatibility genes of specific control has important significance in biology and breeding etc.To screen the genes associated with the self-compatibility of Eruca sative Mill, RNAs isolated from anther and stigma of pre-bloom and after flowering were used to screen differential gene expression in SC and SI of Eruca sative Mill using differential display reverse transcriptase polymerase chain reaction（DDRT-PCR） of fourteen pairs of primers. Then, to analyze the mechanism of related genes in Eruca sative Mill Selfcompatibility, the expression pattern were analyzed of some fragments. In addition, the full-length c DNA were cloned of pectate lyase using RACE technology, and constructed plant expression vector.The main results and conclusions are summarized as follows:（1） There are 11 differential genes were screened between SI and SC in Eruca sative Mill. Including xyloglucan galactosyltransferase, chaperone protein dna J, pectate lyase family protein, 40 S ribosomal protein s19-1, hypothetical protein and so on.（2） The Es PL gene were discovered and cloned in anther of Eruca sative Mill for the first time. The sequence was 1657 bp in length, Containing a ORF of 1371 bp, which encodes a polypeptide of 456 amino acid residues.（3） The predicted protein PI is 9.42, the relative molecular weight is 51.179 kD of Es PL gene. Es PL is a hydrophilic protein has signal-peptide. The prediction of the second structures indicated the Es PL contains 24.56% alpha helix, 29.61% extended strand, 13.16% beta turn and 32.68% random coil. Phylogram tree show that there were closest evolutionary relationship of Brassica napus（ CDY09356.1）, Brassica rape（XP₀09129128.1）and Eruca sative Mill Es PL gene.（4） The expression vector of Eruca sative Mill Es PL gene were constructed successful, which laid the foundation for further study the function of this gene.（5） The DnaJ homolog Gene es AtJ3 were discovered and cloned in stigma of Eruca sative Mill for the first time. The sequence contains a ORF of 297 bp, which encode 98 amino acid residues and has a Dna J-C conserved domain belonged to the plant Dna J superfamily. The es At J3 had no signal peptide and was a hydrophilic protein; The prediction of the second structures indicated the es At J3 was mainly consisted of random coils and α-helices. Prokaryotic expression vector p ET32a- es At J3 was constructed and transformated into E. coli BL21（DE3）.Recombinant es At J3 protein was obtained after induction by IPTG and SDS-PAGE analysis with a MW of 31 k D.（6） The expression pattern analysis showed that the expressions of xyloglucan galactosyltransferase gene in SC stigma were the highest before flowering, The expressions of hypothetical protein SC8 gene in SC anther were the highest after flowering, es At J3 gene expression levels is highest in stigma of pre-bloom of SC and Es PL gene expression levels in SC anther is significant higher than SI anther after flowering. The result indicated that those differential genes might play a role in SI and SC Characteristics of regulation of Eruca sative Mill.