Analysis Of Stress Response Mechanism Of Cadmium Stress Mediated By TFⅢA Zinc Finger Protein GhZAT5-like In Cotton

Soil heavy metal pollution has been a global problem endangering crop growth and human life and health.Therefore,exploring the response mechanism of plants to heavy metal stress has important theoretical significance and application value to solve the problem of heavy metal pollution.In this study,the GhZAT5-like gene cloned from cotton was used to study its response mechanism to cadmium stress.The main results are as follows:1.A total of 343 C2H2 zinc finger protein members were identified in upland cotton genome.Cluster analysis showed that they could be divided into 4 subclasses and clustered into 13 subsets,of which GhZAT5-like gene was located in subclass II.The promoter sequence of C2H2 zinc finger protein gene in upland cotton contains a large number of abscisic acid(ABA),jasmonic acid(MeJA),gibberellin(GA)and salicylic acid(SA)response elements,and ABA and MeJA response elements are the most abundant.2.The GhZAT5-like gene can be induced by cadmium stress and salt stress.With the extension of cadmium treatment and salt treatment,the relative expression of GhZAT5-like gene increased continuously.3.The results of tissue-specific expression analysis of GhZAT5-like gene showed that the expression of GhZAT5-like gene was the highest in cotton leaves,followed by stems,and less in roots.GhZAT5-like is located in the nucleus.4.The analysis of cis acting elements of GhZAT5-like promoter showed that the promoter region of the gene included 7 heavy metal response elements(MER like),7 copper ion response elements(Cu RE),1 drought inducing element(MBS),1 gibberellin response element(P-box),1 abscisic acid response element(ABRE),1 methyl jasmonate response element(TGACG-motif),One element related to meristem expression(CAT-box)and one element involved in seed specific regulation(RY-element).The results of histochemical staining of GhZAT5-like promoter activity in cadmium treated seedlings showed that the transient expression of GhZAT5-like::GUS Arabidopsis seedlings showed stronger GUS staining under cadmium treatment than under the control condition.The 1000-1500 bp promoter fragment was the key fragment leading to the tissue-specific expression of GhZAT5-like gene.5.The overexpression of GhZAT5-like in transgenic lines reduced the tolerance to cadmium stress.Cadmium stress could significantly inhibit the growth and development of transgenic lines and WT plants.Under cadmium stress,the germination time of transgenic lines was longer than WT,the root length was shorter than WT,and the cadmium content was higher than wt.After 12 hours of cadmium treatment,the cat activity of the transgenic lines was higher than that of WT,the POD activity was lower than WT,and the SOD activity had no significant difference with WT,indicating that GhZAT5-like could not regulate the tolerance of plants to cadmium by regulating the activity of antioxidant enzymes.After 12 hours of cadmium treatment,the soluble sugar and proline content of transgenic lines and the expression of auxin polar transport related genes PIN1,PIN3 and PIN7 were significantly lower than those of wt.The results showed that the overexpression of GhZAT5-like could increase the accumulation of cadmium,reduce the regulation of osmotic pressure and interfere with the internal environment stability of auxin,so as to reduce the tolerance of transgenic lines to cadmium stress.6.Using WT treated with cadmium as the control,the transcriptome sequencing of GhZAT5-like overexpression lines treated with cadmium was analyzed.The results showed that a total of 495 differentially expressed genes were found in the overexpression lines.The go enrichment analysis of differentially expressed up-regulated genes was mainly concentrated in the biological processes such as metal ion transport and chelation,reaction to oxygen and nitrogen-containing substances,and protein ubiquitination;Go enrichment analysis of differentially expressed down-regulated genes is mainly concentrated in biological processes and molecular functions such as response to external stimuli such as strong light and high temperature,response to compounds,diacylglycerol o-acyltransferase activity and so on.It shows that GhZAT5-like gene may be involved in a variety of stress response processes in plants.KEGG pathway enrichment analysis mainly includes plant response pathways to stress(biosynthesis pathways such as glucosinolate and cutin;interaction of plant pathogens;metabolism pathways such as sulfur and tryptophan)and photosynthesis(biosynthesis of carotenoids,circadian rhythm-plant).The genes that may be involved in the response mechanism of cadmium stress were selected from the differentially expressed genes,such as INT2,afp1,saur4,ga2ox2,etc.the QRT PCR results showed that the experimental results were basically consistent with the sequencing results,indicating that GhZAT5-like responded to cadmium stress by up regulating the expression of gibberellin and auxin related genes and down regulating the expression of sugar transport and abscisic acid related genes.