Cloning And Expression Of Heavy Metal ATPases Gene HMA2 And Genetic Transformation Of RNAi In Iris Lactea

Iris lactea is one kind of perennial herb plant of Iris of Iridaceae,its place of growth is northwest,northeast,north and other places of China.Its roots are developed and leaf quantity,which has strong adaptability to environment,strong growth and low maintenance cost,and is an excellent ornamental ground of heavy metals tolerance,drought resistance and saline-alkali tolerance.Using Iris lacteal as the research material,the full-length cDNA sequence of HMA2 was obtained by RACE technique.The expression levels of HMA2 in shoots and roots was quantificationally analyzed using real-time RT-PCR technique and the patterns change of Cd2+ accumulation in shoots and roots by using an atomic absorption spectrophotometer was also investigated under various CdCl2·2.5H2O treatments.Total RNA was extracted from the roots of I.lactea to obtain a fragment of IlHMA2 by RT-PCR.Then RNAi expression vector pARTG1G2 with inverted repeats of HMA2 driven by the promoter CaMV35S was constructed based on the intermediate vector pHANNIBAL and the plant expression vector pART27 by restriction enzyme digestion and ligation.Moreover,the recombinant plasmid was transformed into agrobacterium GV3101 by freezing and thawing method,and then was transformed into I.lactea to obtain IIHMA2-RNAi lines.This would be a good basis for clarifying the IIHMA2 mediated Cd2+ long-distance transport in the adaptation of I.lactea to Cd2+ contaminated environments.The results obtained are as follows:(1)We cloned HMA2 from I.lactea,The isolated cDNA was comprised of 3538 bp containing 29 bp 5'and 680 bp 3' untranslated region(UTR)sequence and a 2829 bp open reading frame(ORF).The ORF encodes 942 deduced amino acid residues with estimated molecular mass of 101.5 kDa and a theoretical isoelectric point of 8.12.It shared higher similarity with the others(over 60%).The results showed that the homology of I.lactea compared with Triticum aestivum Linn,Oryza sativa and Arabidopsis thaliana were 68%,66%and 55%,respectively.IlHMA has 8 transmembrane domains,and contains heavy metal transporting ATPase specific motif of GICCPSE?CPC?DKTGT?HP and GDGMNDAPAL.Therefore,HMA2 belongs to the family of heavy metals.(2)The expression of HMA2 with the decrease tendency was mainly in roots at the different concentration of CdCl2·2.5H2O treatment,and The expression abundance of HMA2 showed the decrease tendency with the increase tendency of concentrations(5-150 mg L-1),The expression of HMA2 was significantly higher in roots than in shoot.Under high and low concentration of CdCl2·2.5H2O treatment,the expression of HMA2 with the increase tendency in the shoot and root for the increase of hour(3-168 h),the expression of HMA2 was higher in roots than in shoot and had no significantly effort in shoot.However,the expression of HMA2 with low concentration of CdCl2·2.5H2O treatment was 3.6-2.4 times than that of high concentration in the shoot,and the expression of HMA2 with high concentration of CdCl2·2.5H2O treatment was 3.4-3.3 times than that of low concentration in the root.Then,the expression of HMA2 was induced and regulated by Cd2+ stress Under CdCl2·2.5H2O treatment.(3)It have successful constructed pART27G1G2,and then is transformed into the I.lactea by Agrobacterium injection method,finally screening for To generation of the transgenic I.lactea lines by PCR.According to the statistical results of this study,the plant conversion rate of I.lactea was 5%,indicating that the conversion method is suitable for monocotyledon plants.(4)The IlHM42-RNAi lines was got by way of asexual reproduction.The results showed that IlHMA2 was mainly expressed in roots and induced by Cd2+ stress.Based on the above results,we screened the transformed IIHMA2-RNAi plants with 100 mg L-1 CdCl2 2.5H2O for the treatment of 48 h,and extracted the total RNA of roots.The gene silencing effect was analyzed by semi quantitative RT-PCR.The results showed that compared with the wild type,the expression abundance of IlHMA2-RNAi transgenic lines was reduced in different degrees.Among them,The number of L1 expression abundance of IIHMA2 was down regulated by 30%.However,the expression of L9 and L12 was the most significant decrease in expression abundance,with 88%and 76%respectively.These results indicate that RNA interference has a better silencing effect on target gene.Therefore,Next,the physiological indexes such as Cd2+,Zn2+ concentration,MDA content,REC,chlorophyll content,photosynthetic rate and others of L1,L9 number and L12 number selected of plants in each tissue were determined.This would be a good basis for clarifying the IIHMA2 mediated Cd2+ long-distance transport in the adaptation of I.lactea to Cd2+ contaminated environments.