| Plants grow in an open environment,they will inevitably be affected by various abiotic stresses,such as drought,heavy metals,extreme temperature and salt stress,which seriously affect the normal growth and development.In the process of long-term evolution and adaptation,in order to cope with these stresses,plants have formed a variety of regulatory mechanisms to respond to stresses.PLC(Plantacyanin),one of the blue copper protein familys,plays an important role in plant growth and development as well as in the process of resisting stress.Tea plant[Camellia sinensis(L.)O.Kuntze]is an important economic crop.It has been planted for thousands of years in China.Using ’Longjingchangye’ as a research material,the full-length cDNAs of CsPLC-1,CsPLC-2 and CsPLC-3 genes were cloned,and studied the role and function of CsPLC-3gene under abiotic stresses.The specific research contents and results are as follows:1.To study the effect of copper and aluminum on the growth and development of tea plants,Cu2+(5mg/L,40mg/L)and Al3+(0.5mM,5 mM)were used to treat’Longjingchangye’ tea plants.The contents of Cu2+,Al3+ and the main secondary metabolites including catechins,caffeine and amino acids were investigated.The results showed that the content of Cu2+ in the roots,stems and leaves of tea plants increased with the increase of copper concentration,and the changes of the roots and leaves were also the same with Al3+treatment,but there was no significant change in the content of Al3+in the stem under 0.5 mM,5 mM Al3+ treatment.In addition,the distribution of Cu2+ and Al3+ in tea plant is root>leaf>stem;under Cu2+ and Al3+ treatment,the content of catechin and caffeine decreased,while the content of amino acid increased.2.According to the sequence ofCsPLCgene selected from tea plant genome,we designed the specific primer cloned the CsPLC-1、CsPLC-2 and CsPLC-3 gene.The length were 387bp,396bp and 372bp respectively.The structural analysis of intron exons showed that both CsPLC-1,CsPLC-2 and CsPLC-3 contained only one intron and two exons,and the intron length of CsPLC-3 was longer than that of CsPLC-1 and CsPLC-2.The sequence analysis showed that the sequence similarity of CsPLC-1,CsPLC-2 and CsPLC-3 was low,but all of them contained four conserved structural sites.Phylogenetic analysis showed that CsPLC-1,CsPLC-2 and CsPLC-3 were divided into two Group,in which both CsPLC-1 and CsPLC-2 belonged to Group I and CsPLC-3 belonged to Group II;Promoter cis-acting element analysis showed that CsPLC-1,CsPLC-2 and CsPLC-3gene play an important role in stress/defense,hormone response and growth/development correlation.In addition,the CsPLC-3 gene has the largest number of metal response-related elements.qRT-PCR results showed that CsPLC-1 was highly expressed in root,CsPLC-2was highly expressed in stem,and CsPLC-3 was highly expressed in leaf.3.The expression level of CsPLC-3gene under different abiotic stresses(high temperature,low temperature,drought,salt,Cu2+ and Al3+)was studied,the expression of CsPLC-3 gene increased under salt,Cu2+and Al3+stress,and decreased under drought,under low temperature stress,the expression level first increased and then decreased,while under high temperature stress,it first decreased and then increased,decreased later.4.We constructed the fusion expression vector pCAMBIA1250-CsPLC-3-GFP.The target gene was located on the plasma membrane of onion and tobacco by subcellular localization.In addition,we constructed the yeast expression vector of pDR196-CsPLC,transformed it into yeast by LiAc/PEG method and analyzed the tolerance of yeast to salt,Cu2+ and Al3+,the results showed that CsPLC-3 gene could improve the function of tolerance to salt,Cu2+and Al3+in yeast,and both transgenic yeast and wild-type yeast grow at pH 4/6 better than pH 8.5.The plant expression vector pCAMBIA3320Y-CsPLC-3,which constructed CsPLC-3 gene was introduced into Arabidopsis thaliana by inflorescences disseminated method.Four Arabidopsis transgenic plants were obtained after antibiotics screening. |
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