Selection Of Reference Genes For RT-qPCR And Expression Of Genes Involved In Homoglutathione Anabolism In Soybean Under Phophorus Deficiency

Phosphorus is one of the essential elements of plants,and phosphorus deficiency affects plant growth.Glutathione(Glutathione,GSH)is a ubiquitous antioxidant in plants,which plays an important role in plant resistance to low phosphorus stress.GSH protects plants from the damage of reactive oxygen generated in plant physiological and biochemical activities by the Asada-Halliwell way.GSH is synthesized by the catalytic action of ?-glutamicylated cysteine synthase(?-ECS)and glutathione synthetase(GSHS),y-ECS and hGSHS are respectively encoded by y-ECS and hGSHS.There are regulated relationship during the expression of y-ECS and GSHS,the enzyme activity y-ECS and GSHS,and concentration of GSH in plants under stress.Phytochelatins(PC)are synthesized with GSH as the substrate.PCs are induced by the external heavy metal ions,and they can chelate with the heavy metal ions by the sulfenyl on the Cys to reduce toxicity to the plant.hGSH(Homoglutataione,hGSH)has almost the functions as GSH in soybean,Similarly,hPC(Homophytochelatins,hPC)is almost as PC.Screening of stable internal reference genes is a prerequisite for real-time quantitative PCR(RT-qPCR).In this study,the soybean variety Guixia No.2(GX2),which exhibit stronger resistance to phosphorus deficiency,and the contrasting soybean variety Guixiang No.1(GX1)were cultivated in 1/5 Hoagland contained with full nutrient solution and low phosphorus(0.2 ?mol/L KH2PO4).Root and leaves in process 2 d,4 d,8 d,12 d,and 16 d were collected as material.The stability of 15 reference genes(18sRNA,ACT,ATP,CYP,Ef1-a,Ef1-?,G6PDH,PE,PP2A,PSC,TIF,TUB,UNK1,UNK2 and Letin)was analyzed by GeNorm,NormFinder,BestKeeper,?Ct comparision and RefFinder under low phosphorus stress and the most stable reference genes were used to normalize the expression of y-ECS and hGSHS in different low phosphorus stress periods.The enzyme activity of y-ECS and hGSHS,the change content of hGSH and hPCn(n=2?11)were also detected in low phosphorus stress.After RefFinder comprehensive analysising,the most stable genes in all samples were PSC,18sRNA and TUB,the worst were PE and CYP;the best was PP2A in the low phosphorus treatment while ACT in CK;TIF was the most stable at root and PE at leaves.According to the analysis of pairwise varition,the relative expression of y-ECS and hGSHS should be normalized by three reference genes(PSC,18sRNA and TUB).In the low p efficient soybean variety GX1,the expression fold changes(FC,as compared to normal)of ?-ECS and hGSHS in roots and leaves decreased with the increase of stress duration;while in the high P efficient variety GX2,the FCs of y-ECS in roots and leaves,as well as hGSHS in roots increased with the increase of stress duration;except that hGSHS in leaves decreased.The enzyme activity of y-ECS and hGSH in the roots and leaves of GX2 increased with time,and the increase of root was greater than the increase of leaves,GX1 showed decrease.The concentration of hGSH increased with time both in the roots and leaves tissue of GX2,while GX1 showed the opposite trend.The concentration of hPCs in root and leaves of two soybean varietes was little under low phosphorus stress.In conclusion,the increase enzyme activity of y-ECS and hGSHS and hGSH concentration increasing were molecular mechanism to resistance the low phosphorus stress in soybean;there was a regulated relationship between the expression of y-ECS and hGSH genes and the concentration of hGSH;the phosphorus efficiency was different with variety in soybean.