The Analysis Of Differentially Expressed Gene Rsk Of Brassica Napus Induced By S.Sclerotiorum

The study is based on the significant differences between ZhongR888(Brassicanapus) and Zhongyou821(Brassica napus) on the Sclerotinia scIerotiorum resistanceto establish zhongyou821xzhongR-888 BC₅ near isogenic lines with zhongR888 asthe donor parent and zhongyou821 as the recurrent parent. With the NILs as thematerials, the full-length cDNA of one of the differential TDFs (Transcripts-derivedFragments) related to Sclertiorum resistance was cloned, and the expressions of theTDF between the NILs at different developmental stages and different tissues wereanalyzed. The main results were as follows:The differential TDFs related to Sclertiorum resistance were screened andidentified by means of cDNA-AFLP method. By BLAST analysis of one effectivedifferential TDF called 40-2, it was found that the gene expression products of it waspresumably belong to the leucine-rich repeat protein family. The effective differentialTDF was then amplified using RT-RACE method and the full-length cDNA of 1707bpwith the integrated Poly(A) tail at the 3' end and the untranslated region at the 5' endwere gained. The sequence of the full-length cDNA was logged in Genbank, and theaccession number was DQ983911.Through BLASTx analysis, it was found that the sequence had high similaritywith the mRNA of the disease resistance/leucine rich repeat protein family inArabidopsis, and the value is 87%. Through sequence similarity analysis, it was alsofound that the expression products of the differential gene had a high similarity with akinase/protein binding protein in Arabidopsis, and had a conserved LRR-RI domain,indicating that the differential gene products might be one of the members of theLeucine-rich repeats, ribonuclease inhibitor-like subfamily. So far, there had not anyreport related or similar to this gene or protein in Brassica napus.With the full-length cDNA sequence of the differential TDF 40-2 as basis todesign primers, the genome of zhongyou821 as PCR amplification template, thefull-length of 40-2 genome was got successfully. Sequence measurement showed thatthe genome had a full-length of 1685bp with a coding region between 264 and 1586bp,without any intron sequences. And the accession number of this gene named Rsk onGenBank was DQ983911.The open reading frame of it was between264bp and 1586bp, encoding a proteincomposed of 440 amino acids. The accession number of the encoded protein onGenbank was ABI99471. The protein was made up of 20 familiar amino acids among which leucine had the most content. The MW of the protein was 48.308KD, PI was5.35, so it was an acidic protein. The both two ends of the protein was hydrophilic, thecentre of it was hydrophobic, and the protein had a transmembrane domain.Secondary structure prediction showed that the protein had a majority of random coilstructure, some alpha helix and extended strand structures. Between the 290 and the313 amino acids, there was a LRR domain. Near the C-end of the protein, there was atransmembrane region, and the C-end of it was in the cytosol. It could be predictedthat the protein be located on the cell membrane.With the genome of zhongyou821×zhongR-888 BC₅ as PCR amplificationtemplate, under the same condition with the above, the genome DNA with the fulllength of 1703bp was also got. It was predicted as one gene with an extron between264 and 1604bp coding region, without an intron. And the accession number in theGenbank was EF623492. Sequence alignment showed that the DNA sequencesamplified from zhongyou821×zhongR-888 BC₅ was the same as the Rsk gene fromzhongyou821 in the region between 1 and 1577bp, 1596 and 1703bp, but the 18bpbetween 1578 and 1595bp was absent in the latter. It was presumed that the base pairabsence in the gene level was likely to be related to the differences between thedisease resistances to Sclerotinia sclerotiorum in NILs.The cloned gene from zhongyou821×zhongR-888 BC₅ was presumed to encode446 amino acids. And the the amino acids were the same, except that the six aminoacids between 439 and 444 were absent in zhongyou821. This result corresponded tothe DNA sequence alignment result. It was presumed that the differences might be theheredity foundation that caused the differential expression in NILs induced bySclerotinia sclerotiorum.The results showed that the Rsk gene was expressed a certain quantity in breedson different Sclerotinia sclerotiorum resistance and in leaves and flowers of NILs atdifferent developing stages including seedling period, flower-budding period andflowering period, but there was no evident expression differences betweenzhongyou821 and zhongyou821×zhongR-888 BC₅. It was also showed that the geneexpressed most in flowers at flowering period, expressed lower in leaves of seedlingand flower-budding period.