Gene Cloning And Protein Expression Of Growth Hormone Receptor In Echinochloa Crus-galli Var.zelayensis

In rice fields,Echinochloa crus-galli var.zelayensis is one of the malignant weeds.In recent years,its resistance to quinclorac has become more and more obvious.The mechanims of the resistance to quinclorac in Echinochloa crus-galli var.zelayensis needs to be solved urgently.At present,the researches mainly focused on the downstream hormone response of plant growth hormone biological pathway interfered by hormonal herbicide quinclorac.while the upstream of plant growth hormone biological pathway,there are few studies on the relationship between hormonal balance,hormonal signal perception,signal transduction and quinclorac resistance.As for how quinclorac affects the growth hormone pathway in Echinochloa crus-galli var.zelayensis,there have been no reports.It has been reported that Arabidopsis mutant experiments have demonstrated that AFBs protein mutant plants exhibit varying degrees of auxin response defects.The auxin binding assay showed that the protein of AFBs family is required for auxin binding.Therefore,it is speculated that the AFBs family protein is also likely to be a receptor for plant growth hormone.In this study,we studied Echinochloa crus-galli var.zelayensis and speculated that the growth hormone receptor in Echinochloa crus-galli var.zelayensis might be related to the resistance of Echinochloa crus-galli var.zelayensis to quinclorac.Specifically,the AFBs family proteins mutations in the resistant biotype Echinochloa crus-galli var.zelayensis may cause delay or pause to the hormonal herbicide quinclorac,which may result in hindered signal transduction,and eventually the plant exhibits a drug-resistant phenotype.It is hypothesized that the drug-resistant of Echinochloa crus-galli var.zelayensis is similar to drug-resistant of the AFBs protein mutations in Arabidopsis thaliana,which causes different degrees of auxin response defects in Echinochloa crus-galli var.zelayensis,and further exploration is needed.In this paper,the full-length AFB2 and AFB3 genes sequence in Echinochloa crus-galli var.zelayensis was obtained by gene cloning,and the genes were analyzed by bioinformatics.The open reading frame of EczAFB2 gene is 1728 bp,which can translate 575 amino acids,and the theoretical isoelectric point(pI):7.05.The molecular weight is 64.47kD,the amino acid sequence encoded by EczAFB2 gene is 84%similar to the OsAFB2 gene of the known model plant rice;the open reading frame of EczAFB3 gene is 1722 bp,which can translate 573 amino acids,the theoretical isoelectric point(pI):8.94,molecular weight 64.65 kD,the amino acid sequence encoded by EczAFB3 gene has a similarity of 63%compared to the ZmAFB3 gene of the known plant maize.By gene conserved motif analysis,EczAFB2 and EczAFB3 have three typical conserved motifs,Motif 1,Motif 2 and Motif 3,respectively.By the sensitivity and resistance biotypes of this gene,the first EczAFB2 gene is known.The 56-position leucine(Leu)of the conserved Motif 1(from the initiation codon)was mutated to valine(Val);the conserved Motif 2 interval was not mutated;whereas in the conserved Motif 3,The glutamic acid(Gln)at position 451 was mutated to lysine(Lys)and the histidine(His)at position 452 was mutated to glutamate(Gln);while the EczAFB3 gene was not mutated in the conserved motif.Through structural analysis and protein subcellular localization prediction of EczAFB2 and EczAFB3 genes,it is known that they have a typical F-box domain and a repeating LRRs domain,which are located in the nucleus and have the function of growth hormone signal recognition.According to the prediction of protein tertiary structure,the proteins encoded by EczAFB2 and EczAFB3 genes are all signaling proteins,and the similarity of the pattern plant Arabidopsis AtTIR1 tertiary structure model is used as a reference.The results show that the similarity of EczAFB2 is 61.70%,the similarity of OsAFB2 is 62.23%,the similarity of AtAFB2 is 61.59%,the similarity of EczAFB3 is 55.44%,the similarity of ZmAFB3 is 60.04%,The similarity of AtAFB3 is 60.35%.To further validate the proposed hypothesis,we established the eukaryotic protein expression system of the EczAFB2 gene.The baculovirus expression vector was constructed by using the insect cell baculovirus expression system in the eukaryotic protein expression system,and the EczAFB2 protein was successfully expressed in the insect cell SF9.The results showed that the expressed target protein size was about 64KDa,which was consistent with the theoretical prediction.The technical basis for the interaction between protein and small molecule in vitro to clarify the target receptor of the hormonal herbicide quinclorac in Echinochloa crus-galli var.zelayensis and further reveal whether the quinclorac resistance is related to the hormone receptor.