Cloning And Expression Analysis Of Chalcone Synthase Gene In Loropetalum Chinense Var.Rubrum

Loropetalum chinense var.rubrum belongs to the Hamamelidaceae Rubrum,which has been widely used as color-carving,color-block,shrub landscape,potted landscape plants,or configuration with other ornamental plants,for the prominence of ornamental characteristics,adaptability,stress resistance,strong resistance to cutting,strong plasticity,easy propagation.Since it was found in Hunan in the eighty's of the last century,Its introduction,cultivation,and application by gardeners once led to the rapid development of economy of Liu yang City,Hunan Province,being awarded the honor of "Loropetalum chinense var.rubrum town".And,it becomes one of the main color-leafed plants along the middle and lower reaches of Yangtze River.During the period of it,a number of different varieties of leaf shape,leaf color,flower color or flower shape and other variation types were bred.But for the weak fundamental research,breeding of L.chinense var.rubrum depended on clonal selection.Genetic improvement and germplasm enhancement is slow,the variety is still relatively simple,and the main color of the leaf is red,which is out of the demanding of the market.Therefore,it is necessary for us to carry out explanation on genetic improvement and mechanism of leaf color,which can lay a solid foundation for leaf-color genetic improvement or artificial regulation of L.chinense var.rubrum with modern molecular technology.CHS is the first key enzyme in secondary metabolism of flavonoids and anthocyanin glycosides,plays an important role in this metabolic pathway,expression,CHS gene silence,over-expression or mutation will affect the process directly or indirectly,thus affecting the total output of flavonoid and anthocyanin.Though,"Double Red" L.chinense var.rubrum cultivars "Dayehong" was used as material in this study,using the method of RT-PCR,to investigate the cloning and expression of CHS gene,which will lay a solid foundation for us to do some more research in the genetic improvement and molecular regulation of it.Access to the key findings is as follows:(1)A reliable and effective system of RNA extraction from L.chinense var.rubrum for RT-PCR analysis was developed.The studies were made on RNA of L.chinense var.rubrum obtained from the young leaves of cutting seedling or tissue culture seedling.By using the improved methods of CTAB,the improved methods of SDS,the Trizol method,or the method of RNAplant plus Reagent.The result shows that RNA cann't be obtained by the improved method of SDS or the method of Trizol.It can be obtained by the improved methods of CTAB,but the purity and molecule weight is not good enough for follow-up experiments.Only by using the method of RNAplant plus Reagent can the high molecule weight and high purity RNA be obtained from the young leaves steadily.(2)The gene CHS sequence of the leaf color appearance was cloned from the young leaves of the L.chinense var.rubrum 'DayeHong',which is 927bp,encoding 232 amino acids,named "LcvrCHS1"(GenBank accession number:JQ609678).(3)By comparing its sequence with homologues from other plants by BLASTn,we concluded that CHS gene had logged a walnut,Camellia CHS sequence homology was 83%,and other plants(Hydrangea,grape,peach,potato,licorice,euptelea)CHS sequence can reach more than 80%.The amino acid sequence of the deduced amino acid sequence had the same highly homologous with amino acids encoded by CHS of camellia,avocado,grape,pear,pear,red azalea,which is 98%.After being tested with the Signal P3.0 Server,the result showed that LcvrCHS1 protein does not contain any signal peptide sequence,belonging to the non-secretory protein.(4)Detection and analysis of CHS gene expression and leaf color phenotype of tissue culture seedling of L.chinense var.rubrum that had been treated by the temperature of 30?/22.7 ?(day/night),35 ?/35 ?(day/night),40 ?/35?(day/night),15.3 ?/8.3 ?(day/night)a week shows that,the leaf color s reviving obviously being treated with isothermal treating temperature to 35?,leaf color is remaining basically in 15.3 ?/8.3?(day/night),treatment with 30 ?/22.7 ?(day/night)is willing to revive weakly.At the same time,the results showed that the CHS gene is almost no expression with treatment of isothermal treating temperature to 35 ?,is weakly expressed in 30 ?/22.7 ?(day/night)treatment,while the expression decreased significantly at the condition of 15.3 ?/8.3 ?(day/night).We can concluded that the main inducing factor of leaf reviving is the treatment with the high temperature of 35?,and we can indicate that the reason may be related to CHS gene was no expressed.