| LEA(Late embryogenesis abundant)proteins,a kind of stress-related protein,are involved in plant responses to different abiotic stresses.In this study,5 potential LEA gene family members were identified from Brachymenium exile.LEA proteins were classified into 3 groups,including LEA2,LEA5 and SMP subfamilies.There are 3 LEA5 subfamily genes,named Be LEA5-1,Be LEA5-2,and Be LEA5-3,respectively,and LEA2 and SMP subfamily,named Be LEA2 and Be SMP,respectively.Full sequences of LEA family members were cloned via PCR.The length of LEA gene full sequences was between 428 bp and 1792 bp and the number of introns was 1-3.The length range of open reading frames was between 267 and 915 bp,encoding 88-304 amino acids.The theoretical isoelectric points of the LEA gene family members were between 4.26 and 6.53,and the molecular weights were between 9148.91 and 31132.69 Da.The instability indices were between 44.67-101.32.The instability indices ranged from 17.01 to 42.35,which Be LEA5-1,Be LEA5-2,Be LEA5-3 and Be LEA2 were stable proteins,while Be SMP was unstable protein.All grand average of hydropathicity indices were negative,indicated that LEA gene family members were hydrophilic proteins.The LEA family gene family member proteins were mainly composed of Alpha helix,random coil,beta turn and extended strand.Alpha helix and random coil had the highest proportions,accounting for 66.22%-87.78%.The prediction of the 3D structure of LEA family members showed that the number of α-helices was 1-2,and the number of β-sheets was 1-10.Be LEA2 had the most complex structure.Promoter sequences of LEA family members were cloned via Hi Tail-PCR.The results of Plant CARE analysis showed that the promoters of Be LEA5-1,Be LEA5-2,Be LEA2 and Be SMP genes all had basic elements such as TATA-box and CAAT-box(Be LEA5-1 did not contain TATA-box).In addition,a large number of elements related to the regulation of abiotic stress were also found,mainly including abscisic acid response elements such as ABRE,MYB and MYC;methyl jasmonate response elements such as CGTCA-motif,TGACG-motif and G-box.Growth hormone response elements such as Aux RR-core;cis-acting elements related to salicylic acid response such as TCA-element;regulatory elements for defense gene activation such as MBS,TC-rich repeats and W box elements;light-responsive elements such as Sp1.Fluorescence quantitative analysis of LEA family members by q PCR showed that the expression of Be LEA5-2,Be LEA5-3 and Be LEA2 in the sporophyte of the asexual generation,the expression of Be LEA5-3,Be LEA2 and Be SMP in gametocytes of asexual generation,and the expression of Be LEA5-2 and Be LEA5-3 in gametocytes of sexual generation were significantly increased under dehydration stress,while the expression of Be SMP in the sporophyte of asexual generation,and the expression of Be LEA2 and Be SMP in gametophytes of sexual generation were significantly decreased.The recombinant vector(p Thiohis A/Be LEA5-1)was successfully constructed by homologous recombination,and a recombinant strain(BL 21/Be LEA5-1)was obtained.The results of SDS-PAGE showed that the Be LEA5-1 protein of the recombinant strain(BL 21/Be LEA5-1)was expressed,and the expression increased with the increase of induction time.The results of salt tolerance analysis showed that the expression of Be LEA5-1 protein increased the tolerance of recombinant strain(BL 21/Be LEA5-1)to salt stress(500 mmol / L Na Cl and 700 mmol / L KCl).In conclusion,the members of the LEA family from Brachymenium exile may have a response mechanism to the regulation of abiotic stress.This study provides a theoretical basis for the functional study of the LEA gene family in mosses,and provides a potential application prospect for molecular breeding to improve salt tolerance of plant. |
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