A Root CDNA Library Construction, Analysis And Root Specific Expressed Genes Cloning From Callerya Speciosa

Callerya speciosa, a medicinal plant of Leguminosae, another name for Millettia Speciosa, its dry root is main raw material for variety of proprietary Chinese medicine and is largely used for medicinal food in Guangdong and Guangxi folk. In the literatures, only the historical background on chemical constituent, potency and crude medicines were reported. So the molecular biology research on root enlargement related of Callerya speciosa should be researched.The swelling characteristics of the Callerya speciosa root is closely related to yield, its swelling properties in the differences between the different Callerya speciosa germplasm. To study the root enlargement related genes of Callerya speciosa, a cDNA library of the tuberous root of Callerya speciosa was constructed, and then, expressed sequence tags (ESTs) were analyzed. According to the results of the analysis of the library, by using semi-quantitative RT-PCR analysis of expression levels in different tissues of Specious millettia tuberous root, leaves, branches, the4specific expression genes in tuberous root were screened. We obtained the full-length cDNA of this4genes by RACE, and did the bioinformatics analysis on the genes. The main results were as follow:1. The total RNA was abstracted with the CTAB method from the tuberous root of Callerya speciosa. And the cDNA library was constructed. By the quality inspection:The titer of the cDNA library was6.17X107pfu/mL, and the rate of recombinant was90.9%,the insert size ranged from1.3kb. Therefore, the cDNA librarie the tuberous root of Millettia Speciosa have been successfully constructed.2.1728clones were chosen randomly from this library, and1571valid expressed sequence tags were generated. Then the ESTs were clustered by CodonCode Aligner software. As a result,1009unigenes were obtained, in which there are236Contigs and773Singletons. By the Blastn-x comparation shows that738unigenes can be did function annotation with Gene Ontology, there are468genes with molecular function. And the remainder269genes are considered to be unknown genes. According to the molecular function of GO,11categories are formed. Higher proportions of genes related to protein binding activity and catalytic activity are40%and33.5%respectively. And structural molecular activity, regulation of enzyme activity and transport activity are11.3%,6.2%and3.8separately. Moreover, percentage of genes relevant to transcription regulator activity, molecular sensing activity, translation regulation activity, antioxidant activity, launch activity and protein lable is less than1%.3. According to the result of the library EST analysis, independent genes was experimented by semi-quantitative RT-PCR, and then get4ESTs of root-specific expression genes. The full-length cDNA of these genes were obtained by RACE.4. Analysis of the4root-specific expressed genes nucleic acid and amino acid were performed with Bioinformatics. The characteristic was obtained, and function was predicted.5. The result of Semi-quantitative RT-PCR show that expression level of8C10gene is higher obviously in the tuberous root;13E07、15A04、15B06genes Just express in the tuberous root. Speculated that the4genes its function may be relate to swelling of the tuberous root.