Expression Studies, Based On The Induction Of Three Functional Genes In Mulberry Cdna Library

Mulberry (Morus alba) is an economically important plant used for sericulture. our country has the most abundant mulberry germplasm resources. The growth and productivity of mulberry is adversely affected by abiotic and biotic stresses. Efforts to investigate the functional genes and their molecular adaptation mechanisms of stresses and to strengthen stress tolerance in this plant are of fundamental importance to the preservation of these genetic resources and to improve the yield of mulberry. However, the research of the functional genes from mulberry is in a backward state in comparison with other plants, and the exact function of some functional genes and their encoded proteins in the stress response in mulberry is still not fully understood yet. Therefore, the research of functional genes in mulberry is one urgent and the meaningful work.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 three important genes and carried out the analysis of their sequences. Furthermore, we conducted the functional verification of the three genes by the means of stress-induced method based on the prediction of their functions.The main results were summarized briefly as follows:1,EST encoding Chitinase was isolated from the cDNA library. And it's full open reading frame were obtained by RACE and RT-PCR for the first time. A full length cDNA sequence coding for Chitinase in mulberry was designated M-chitinase (GenBank accession number: HQ117891). Sequence analysis showed that the M-chitinase is 1392 bp long and contains a 60 bp 5'-UTR (untranslated region) and a 255 bp 3'-UTR. It's opening frame(ORF) is of 1077 bp,encoding 358 amino acids with apredicted molecular weight of 38.52KD and an isoelectric point of 4.466. Homology analysis revealed that M-chitinase gene are highly conservative in mulberry and other species including N. khasiana, Zea mays and Zea Diploperennis. Phylogenetic analysis based on M-chitinase gene with other 19 species showed that mulberry shows closer relationship with Nicotiana gossei, nicotiana tabacum, Capsicum annuum and rock cress. The results of semi quantitative RT-PCR analysis showed that the transcriptional level of M-chitinase mRNA in the young leaf. And the transcriptional level of M-chitinase mRNA changed significantly under the conditions of cold, SA and ABA stresses respectively compared to the normal growth environment. 2,EST encoding PGIP was isolated from the cDNA library. And it's full open reading frame were obtained by RACE and RT-PCR for the first time. A full length cDNA sequence coding for PGIP in mulberry was designated M-PGIP (GenBank accession number: HM044383). Sequence analysis showed that the M-PGIP is 1274bp long and contains a 93bp 5'-UTR (untranslated region) and a 179bp 3'-UTR.It's opening frame(ORF) is of 1002bp,encoding 333 amino acids with apredicted molecular weight of 37.29KD and an isoelectric point of 7.25. Homology analysis revealed that M-PGIP gene are highly conservative in mulberry and other species including Prunus americana, Prunus persica and Prunus mume. Phylogenetic analysis based on M-PGIP gene with other 19 species showed that mulberry shows closer relationship with Cucumis melo, Vitis labrusca x Vitis riparia, Vitis vinifera and Actinidia deliciosa. The results of semi quantitative RT-PCR analysis showed that the transcriptional level of M-PGIP mRNA in the young leaf. And the transcriptional level of M-PGIP mRNA changed significantly under the conditions of SA,ABA and salt sresses respectively compared to the normal growth environment.And we had expressed the recombinant protein to lay a good foundation for disease resistance Engineering of transgenic plants.3,The full length cDNA sequence coding for photosynthesis system II PsbR in mulberry Using the expression sequence label (expressed sequence tags, ESTs)was adopted the RT-PCR method to clone ,and named MpsbR (the Genbank accession number:GU937873 ). The sequential analysis indicated that this gene is 623bp long, and contains a 56bp 5 ' - UTR and a 306bp 3 '– UTR.It's opening reading frame (ORF) is of 261bp, encoding 86 amino acids with apredicted molecular weight of 8.95KD and an isoelectric point of 11.09. Homology analysis revealed that , MpsbR gene are highly conservative in mulberry and other species including peanut, soybean and Prunus mume.; The phylogenetic analysis based on MpsbR gene with other 20 species showed that mulberry shows closer relationship with peanut, sea mulberry, pear and soybean. The results of semi quantitative RT-PCR analysis showed that the transcriptional level of MpsbR mRNA in the young leaf and the top bud. Its action mechanism is waiting for further studies.