Isolation And Expression Analysis Of Differential Genes Between Jin Guanyin(Camellia Sinensis)and Its Parents

The generation C.sinensis cv.Jinguanyin,an excellent tea variety,was produced by artificial hybridization between female parent C.sinensis cv.Tieguanyin and male parent C.sinensis cv.Huangdan.As for bud color traits,C.sinensis cv.Jinguanyin has the existence of differences with its parents,and the character of yield,aroma and adaptability is over parents,which showed strong heterosis.The heterosis genetic mechanism of tea plant is currently stay on the aspect of morphological characteristics,physiological and biochemical characteristics,tea quality and so on.The mechanism of tea plant heterosis explained by genetics theory is also rarely reported.To explore the molecular mechanism,genetic regulation and resistance related gene cloning of apparent and inherent differences between C.sinensis cv.Jinguanyin and its parents,it can provide the theoretical basis for the early selection of tea plant crossbreeding,the theory of hybrids formation of tea plants and the analysis of the molecular mechanism of tea plant stress resistance.This research used fresh spring leaves of C.sinensis cv.Jinguanyin,female parent C.sinensis cv.Tieguanyin and male parent C.sinensis cv.Huangdan as the materials,to construct Jinguanyin-Tieguanyin and Jinguanyin-Huangdan two positive DSN mediated suppression subtractive hybridization cDNA libraries.The positive clones of these two positive subtractive libraries were screened by reverse Northern-Blotting technique and sequenced.The results were analyzed by bioinformatics,and their functions were predicted and classified.The quantitative real time PCR technique was used to detect the expression quantity change between C.sinensis cv.Jinguanyin(hybrid)and the differential genes(related to growing development,resistance and quality)of its parents,so as to analyze the expression patterns of differential genes.RT-PCR and quantitative real time PCR were used to validate genes with over parent and lower parent expression patterns.Using the quantitative real time PCR technique to test out the differences in the expression quantity of genes with over parent and lower parent expression patterns in other two generations of C.sinensis cv.Huangguanyin and C.sinensis cv.Jinmeigui,so as to speculate the genotypes of parents with heterosis.The sequence of CsENO gene involved in resistance was screened out from the heterosis genes obtained from the male parent C.sinensis cv.Huangdan and the generation C.sinensis cv.Jinguanyin suppression subtractive hybridization library,and then CsENO was cloned and verified from C.sinensis cv.Tieguanyin.Using bioinformatics software to analyze the sequence and using quantitative real time PCR technique to detect the dynamic expression of this stress resistance gene under four kinds of adversity stress treatments.The results are as follows:1、Construction and screening of two positive suppression subtractive cDNA libraries from C.sinensis cv.Jin Guanyin and its parents:The titer of two subtractive libraries were 2.3 × 105 cfu/mL and 3.5 ×105 cfu/mL respectively.The size of the fragment inserts of two subtractive libraries were both range from 1000 bp to 3000 bp.A total of 90 and 113 single-gene sequences were respectively screened from the Jinguanyin-Tieguanyin and Jinguanyin-Huangdan positive suppression subtractive libraries using reverse Northern-Blotting technique.There were 14 common genes among them,76 differential gene traits tended to Huangdan and 99 differential gene traits tended to Tieguanyin.The genes involved in tea plant growing development,secondary metabolism,aroma metabolism,adversity stress,signal transduction,energy metabolism,biological regulation,molecular functions and cell compositions were all screened from these two positive subtractive cDNA libraries.2、Genetic analysis of differential gene expressions between C.sinensis cv.Jinguanyin and its parents:The genes related to growing development,resistance and quality were all expressed in C.sinensis cv.Jinguanyin and its parents.According to the differences in expression,these genes can be divided into 5 expression patterns.Fifteen genes were randomly selected from the expression patterns of over parents and lower parents.They were detected the differences in their expression levels in C.sinensis cv.Jinguanyin and its parents by RT-PCR and quantitative real time PCR technique.The results showed that the intensity of the bands of fifteen RT-PCR products were consistent with that of quantitative real time PCR and they can be regarded as over parents and lower parents expression feature.According to the difference of quantitative expression among C.sinensis cv.Jinguanyin,C.sinensis cv.Huangguanyin,C.sinensis cv.Jinmeigui and their parents,the fifteen genes can be divided into three types:the expression level of three generations were all higher than their parents(heterosis),were all lower than their parents(hybrids weakness)and some more than their parents,some less than their parents(both heterosis and hybrids weakness).And parental genotypes of each type were different.3、Cloning and verifying a sequence of gene involved in resistance,which was screened out from the heterosis genes:The cDNA length of this sequence,which was named as CsENO(Accession NO.KX962311),was 1 753 bp,containing a 1 332 bp open reading frame(ORF).CsENO sequence encoded 444 amino acid residues.Amino acid sequence analysis indicated that CsENO had the highly conserved regions in plants with specific structure domains of ENO and had the closer genetic relationship with Malus domestica and Pyrus × bretschneideri,which shared 84%identity in amino acids.Bioinformatic prediction showed that CsENO belonged to the stability and hydrophilic protein with no transmembrane structure and signal peptide.It might be localized in other subcells and there were phosphorylation sites within the polypeptide chain.Secondary structure of CsENO was predicted to be mainly consisted of alpha helix.The quantitative real time PCR showed that CsENO was all expressed in response to cold stress,ABA,high salinity as well as drought treatment.And its expression was induced by different degrees.