Molecular Cloning Of Two Functional Genes And A Promoter From Mulberry (Morus Multicaulis), And Patterns Of Genes Expression

Mulberry (Morus L) is an economically important plant used for sericulture.Thousandsyears ago has been used by working people in China，and our country has the most abundantmulberry germplasm resources. But, the research of the functional genes from mulberry is ina backward state in comparison with other plants, the growth and productivity of mulberryis adversely affected by abiotic and biotic stresses. Therefore, the study of some importantfunctional genes and molecular mechanisms under abiotic stress conditions, can enhanceresistance to various stress conditions, to improve the yield of mulberry leaves and mulberrygermplasm preservation of great importance.As a first step towards characterization of functional genes in mulberry, in this paper,we constructed a cDNA library from young mulberry leaves. Based on the research above,we cloned two important genes and carried out the analysis of their sequences. Furthermore,we conducted the functional verification of the two genes by the means of stress-inducedmethod based on the prediction of their functions.The main results were summarized brieflyas follows:1、 Molecular cloning， Sequence analysis and induced expression studies ofphosphatidylinositol(4,5)-bisphosphate gene and promoter from mulberry(Morusmulticaulis).EST encoding phosphatidylinositol(4,5)-bisphosphate was isolated from the cDNAlibrary. And the full open reading frame is obtained by RT-PCR for the first time. A fulllength cDNA sequence coding for phosphatidylinositol(4,5)-bisphosphate in mulberry wasdesignated MmPIP2(GenBank accession number: JN716318). Sequence analysis showedthat the MmPIP2is1079bp long and contains a102bp5’-UTR (untranslated region) and a128bp3’-UTR.It’s open reading frame (ORF) is of849bp, encoding282amino acids withapredicted molecular weight of29.87KD and an isoelectric point of8.91. While use thegenomic DNA of mulberry (Morus multicaulis) as a template, cloned gene promoterfragment of PIP2. The promoter fragment sequence structure analysis by the promotersequence analysis software of PLACE. The results showed that the promoter fragmentcontaining the core promoter sequences, in addition to the typical TATA-box, GATA-boxand CAAT-box and other conservative elements, there are many other important elements,such as the role of AE-box, I-box and ABRE and so on.The results of quantitative PCRanalysis showed that the transcriptional level of MmPIP2mRNA changed significantly under the conditions of hypothermia, arid, drought stress of PEG–6000, salt stress andresistant and susceptible respectively compared to the normal growth environment.2、Sequence analysis and expression of the Ribosomal protein S3a gene, MmRPS3a, inmulberry (Morus multicaulis)A full-length cDNA sequence coding Ribosomal protein S3a of mulberry tree, whichwe designated MmRPS3a, was cloned based on mulberry expressed sequence tags (ESTs).Sequence analysis showed that the MmRPS3a is1089bp long and contains a80bp5’-UTR(untranslated region) and a220bp3’-UTR.It’s open reading frame (ORF) is of789bp,encoding262amino acids with apredicted molecular weight of30.053kD and an isoelectricpoint of9.84. Homology analysis revealed that MmRPS3a gene is highly conservative inmulberry and other species including Morus notabilis, Theobroma cacao and Ricinuscommunis. Phylogenetic analysis based on MmRPS3a gene with other species showed thatmulberry shows closer relationship with Prunus persica, Arabidopsis thaliana，Solanumtuberosum, Solanum lycopersicum and Vitis vinifera.The results of quantitative PCRanalysis showed that the transcriptional level of MmRPS3a mRNA changed significantlyunder the conditions of hypothermia, arid, salt stress and resistant and susceptible. However,the gene has the characteristics to be further studied, because studies of plants to abioticstress adaptation should adopt a multi-factor test analysis.