| Mulberry(Morus L) is important perennial crops and it is the only feed of silkworm. After a long natural and artificial selection, mulberry has forming a rich mulberry germplasm resources. However, compared with other plants that we are in a state of backwardness in terms of mulberry gene function studies. Efforts to investigate the functional genes and their molecular adaptation mechanisms under stresses plays an important roles in saving mulberry resource sand improving production of mulberry.In this research, three significantly different ESTs sequences of mulberry were cloned by suppression subtractive hybridization.The full-length c DNA sequences of two genes were also cloned by RT-PCR and RACE. then Study on the Expression Pattern of Ma-TCTP gene and Ma-CHS3 gene in different stress-induced by q RT-PCR.In addition,The ANS gene sequences from mulberry were cloned and then ligated it into expression vector 1302. It is ready for conducting functional studies on Arabidopsis. The main contents are as follows:(1)Purple and green shoots of mulberry suppression subtractive hybridizationThe color of mulberry shoots is related to Anthocyanin. Mulberry has a lot of genes about anthocyanin synthesis and regulation. We take the mulberry purple and green buds as material, by suppression subtractive hybridization. The hybridization products obtained different band group by agarose gel electrophoresis,it has three significant bands. We were recovered and sequenced, in which 2 bands of EST sequences. The EST sequences obtained by blast analysis and homology analysis, one of which may be related with the anthocyanin.(2)Molcecular cloning and expression pattern of Ma-TCTP gene and Ma-CHS3 gene in different stress-inducedone gene sequence is obtained from the suppression subtractive hybridization and another from mulberry leaves c DNA library is anthocyanin related gene called CHS3. Full length c DNA of these genes named as Ma-TCTP(Gen Bank accession No. KP228013)and Ma-CHS3 were cloned by reverse transcription polymerase chain reaction(RT-PCR) and rapid amplification of c DNA ends(RACE) method. Sequence analysis showed that full length c DNA of Ma-TCTP was 841 bp including 101 bp 5’-UTR and 233 bp 3’-UTR. Its ORF was 507 bp and encodes a protein of 168 amino acids with a calculated molecular weight of 18.7362 KD and predicted isoelectric point of 4.53. Homology analysis showed that, Ma-TCTP gene is highly conserved in all species. Full length c DNA of Ma-CHS3 was 1234 bplong long,consisting of 173 bp 3’untranslated region and1017 bp open reading frame(ORF) which encodes for 338 amino acid. Structural analysis showed that it has a calculated molecular weight of 36.7018 KD and has isoelectric point of 7.58. Homology analysis showed that this gene similarity with the CHS family is very high. Anthocyanin related genes not only color, but also with the relevant plant resistance. Real-time PCR revealed that the transcription level of Ma-TCTP and Ma-CHS3 gene of m RNA in different tissues and low temperature, high temperature stress are different.But the mechanism remains to be further studied.(3)Construction of expression vector of ANS gene of mulberryANS sequence was taken from NCBI, primers were designed with restriction sites. The full-length c DNA sequence of ANS from mulberry were obtained by RT-PCR. the ANS gene was separately, then 1302 expression vector were digested by enzyme, Purification, connection,at last, put the ANS gene into 1302 vetor and finally sent to the sequencing. |
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