| Heavy metal transporting ATPase(HMA)in plants involves the absorption,transport,detoxification and enrichment of heavy metals by plants.In order to explore the role of heavy metal ATPase HMA in the cadmium tolerance mechanism of ramie,this study used homologous cloning to obtain ramie Bn HMA1,and carried out bioinformatics analysis and expression analysis;constructed a yeast cDNA library and successfully constructed p GBKT7-Bn HMA1 fusion vector and p426GPD-Bn HMA1 yeast expression vector,the Cd-resistant phenotype of Bn HMA1 yeast was analyzed,which laid the foundation for the subsequent study of the biological function of Bn HMA1.The main research results are as follows::(1)Design specific primers based on the conservative sequence of the HMA gene in the transcriptome sequencing results of Boehmeria nivea,and use RACE(rapid-amplification of cDNA ends)technology to obtain the full-length HMA cDNA sequence from ramie(Genbank: MK733841.1).The gene has a full-length cDNA of 2 811 bp and contains an open reading frame of 2 487 bp.The encoded protein contains 828 amino acid residues,a molecular weight of 90.70 k Da,and an isoelectric point(p I)of 8.84.It belongs to the P-type ATPase family member.The similarity with Morus notabilis and Pyrus bretschneideri is more than 80%.(2)qRT-PCR analysis of the expression of BnHMA1 in different cadmium-tolerant ramie varieties.The results showed that the relative expression of Bn HMA1 in ramie was leaf>bark>root>bar.The expression of Bn HMA1 in leaves,stems and roots of the cadmium-sensitive variety’Xiangju No.3’before and after cadmium stress was higher than that of the other two varieties.The difference in gene expression may be one of the reasons for the different cadmium tolerance of ramie varieties.(3)Using the SMART cDNA synthesis technology and constructing the ramie cDNA library by comparing the two purification methods,the experimental results show that the segmented gel recovered and purified double-stranded cDNA is better than SPIN+ in removing small fragments,library titer and conversion efficiency TE-400 purified double-stranded cDNA purification method is better,recombination rate,transformation efficiency and library titer are 92%,2.5 × 107cfu/ml,1.32 × 107cfu/m L;SPIN + TE-400 purified double-stranded cDNA purification method The recombination rate,transformation efficiency and library titer were 64%,1.74×106cfu/ug,1.32×107cfu/m L,all of which reached the yeast screening library standard.(4)The pGBKT7-BnHMA1 fusion vector was successfully constructed.In the study of yeast self-activation activity,p GBKT7-Bn HMA1 and the positive control grow in the same way,and both can grow in the defective medium,indicating that the Bn HMA1 protein has the characteristics of transcriptional regulation.It is likely that there is a transcriptional activation domain with transcription factors.Constructed p426 GPD-Bn HMA1 yeast expression vector,transformed into heavy metal-sensitive yeast mutant △yap1 and wild-type Y252,and subjected to heavy metal Cd(0,20,30,50 u M/L)stress treatment to the transgenic yeast By observing the growth status of yeast,it was found that the transgenic yeast showed certain sensitivity to Cd stress. |
PDF Full Text Request