Cloning And Function Analysis Of Heavy Metal ATPase Gene HMA2 In Mulberry

In recent years,the proportion of arable land polluted by heavy metals in China has been increasing year by year.Cd pollution ranks first and seriously threatens the safety of agricultural and forestry production in China.Cd in the soil is easily absorbed by plant roots and enriched in plants,and then enters the human body through the food chain,endangering human life and health.Therefore,developing an efficient remediation technology system for Cd-contaminated soil is of great practical significance.So far,heavy metal contaminated soil remediation techniques such as guest soil method,lime improvement,chemical leaching,heat treatment,and electric repair have been developed.Among them,phytoremediation is a green and sustainable remediation method that can gradually reduce the concentration of heavy metals in contaminated soil through the uptake,migration and accumulation of heavy metals by plants to achieve the purpose of remediation.The effectiveness and efficiency of phytoremediation are severely limited by the slow growth and small total biomass of super-enriched plants that absorb,transport,and accumulate minimal amounts of total heavy metals.Therefore,the use of fast-growing and biomass-rich plants to treat soil heavy metal pollution has attracted extensive attention of scientists.Mulberry(Morus alba L.)is an essential economic tree with a well-developed root system,high biomass,wide distribution,adaptability,perennial,and pruning resistance.At the same time,mulberry leaves are the main feed for domestic silkworms,and the main economic product,silk,is used for weaving rather than consumption.Studies have shown that heavy metal pollution management through sericulture systems has great potential for development by achieving ecological management and economic benefits.Recently,the use of mulberry sericulture system for soil heavy metal pollution management has attracted the interest of environmental pollution researchers and gradually become a hot spot in this field.Heavy metal ATPases(HMA)are proteins that specifically transport heavy metal ions in plants.They play an essential role in the uptake,transport,and compartmentalization of heavy metals in plants.Although the functions of HMA genes have been resolved in many plants,the functional analysis of HMA genes in mulberry has not been reported.The functional analysis of mulberry HMA genes have important theoretical and practical implications for the use of mulberry planting and sericulture systems for soil heavy metal pollution management.Based on the above,this study identified the HMA gene family of mulberry based on the genomic data of Morus alba by bioinformatics,and analyzed its sequence characteristics,phylogenetic tree,promoter elements,etc.;cloned the CDS full-length sequence of MaHMA2 gene,and analyzed the expression changes of MaHMA2 in mulberry under different concentrations of cadmium stress for 24 hours by quantitative fluorescence PCR;the subcellular localization vector was constructed and transiently expressed in tobacco to observe its localization;the yeast heterologous expression vector was constructed to determine its function;the overexpression vector was constructed and heterologously expressed in tobacco mediated by Agrobacterium tumefaciens,and the MaHMA2 overexpression transgenic tobacco strain was successfully obtained and subjected to cadmium stress,and then the accumulation of Cd in transgenic tobacco and the physiological and biochemical indicators were analyzed.The main results are as follows:1.Identification and bioinformatic analysis of the MaHMA gene family.After further analysis of the structural domains and exclusion of genes with significant differences,13 HMA genes were obtained,named MaHMA1 to MaHMA13.Analysis of the gene structures of MaHMA1 to MaHMA13 revealed that MaHMA1 and MaHMA2 had similar structures.A phylogenetic tree was constructed with the rice,Arabidopsis,and poplar HMA proteins family as well as nine HMA proteins that had been reported in the literatures,and the results showed that the 13 MaHMAs were divided into two subfamilies,Group1(transports Zn/Cd/Co/Pb)and Group2(transports Cu/Ag).MaHMA1,MaHMA2,and MaHMA3 were grouped in the same group(Group1),where MaHMA1 and MaHMA2 are very similar in evolutionary distance to rice and Arabidopsis,which have been reported to be involved in cadmium transport.2.Expression Analysis of MaHMA2.To observe whether the expression of MaHMA2 was affected under Cd stress,one-month-old mulberry Guisangyou62 seedlings were treated with different concentrations of Cd under 30 and 100 μM Cd stress,and the change of MaHMA2 expression within 24 h was detected by quantitative fluorescence PCR,and the results showed that MaHMA2 expression was highest in the roots,indicating that the gene mainly plays an important role in the roots.Moreover,it was revealed that the expression of MaHMA2 was inhibited when the Cd concentration in the roots was too high.3.Study on the function of MaHMA2 genes.Tobacco subcellular localization experiments showed that MaHMA2 was located on the cell membrane,indicating that MaHMA2 performs its function on the cell membrane;yeast heterologous expression experiments showed that MaHMA2 can transport Cd into yeast cells and participate in the transport of Cd from extracellular to intracellular;five tobacco positive plants overexpressing MaHMA2 were obtained,and the lines with higher expression and those with lower expression were selected for subsequent experiments.The plants were treated with different concentrations of Cd stress,and their Cd accumulation,physiological and biochemical indicators were analyzed.The results showed that the Cd content in the shoots of the transgenic tobacco was higher than that in the roots,and the Cd concentration in the shoots of the transgenic tobacco line was positively correlated with the expression of MaHMA2,while the opposite was true for the roots;the growth of the transgenic plants was significantly inhibited by the stress treatment,and the Cd concentration in the stress treatment was negatively correlated with fresh weight of the plants;compared with the empty vector,the transgenic lines grow better in the roots and vice versa in the shoots;tobacco plants with higher MaHMA2 expression were less vigorous than those with lower expression;heavy metal stress accelerated leaf aging and the total chlorophyll content of tobacco lines with higher MaHMA2 expression was lower than those with lower expression;the content of malondialdehyde(MDA)increased gradually with increasing concentrations of heavy metal treatments,the MDA content of tobacco lines with higher expression of MaHMA2 was higher than that of lines with lower expression,and vice versa in roots;the overall trend of catalase(CAT)activity and proline content were consistent with the MDA.These results indicated that the overexpression of MaHMA2 enhanced the transport capacity of Cd from roots to shoots.This study completed genome-wide HMA family identification and evolutionary analysis of cultivated mulberry,and the MaHMA2 gene was screened as the target gene for subsequent functional studies.The results demonstrated that the expression of MaHMA2 was regulated by Cd stress,and the gene might play an essential role in the long-distance transport of Cd in mulberry.