基因编辑MeERF127提高木薯抗旱和耐盐性

Gene Editing MeERF127 Enhances Drought and Salt Tolerance in Cassava

干旱、高盐和低温严重损害植物的细胞,抑制其生长,显著降低作物产量。AP2/ERF超家族中的乙烯响应因子(ERF)在植物的生长发育和逆境应答中起关键作用。为了研究MeERF127在木薯响应非生物胁迫中的作用,本研究从木薯中克隆MeERF127基因,对其进行序列比对、亚细胞定位、转录活性分析、表达模式分析,构建MeERF127基因编辑载体获得转基因木薯,对其在干旱、盐和低温胁迫处理后的表型和生理指标进行分析,并且分析胁迫响应基因的表达。结果显示:MeERF127基因CDS全长为711 bp,氨基酸序列的N端和C端分别包含YRG和RAYD元件,第14位和第19位的氨基酸是丙氨酸和天冬氨酸,表明MeERF127属于ERF亚家族;MeERF127蛋白亚细胞定位于细胞核,具有转录因子活性;MeERF127在茎中的表达量最高,在愈伤组织中的表达量最低,在块根形成期(植后80 d)的表达量最高,在干旱和盐胁迫后的表达水平上升,在低温胁迫后略下降;构建基因编辑载体,通过农杆菌侵染木薯愈伤组织,Hi-TOM高通量测序结果表明得到了22个碱基缺失和3个碱基缺失的MeERF127基因编辑株系;在干旱和盐胁迫处理后,基因编辑木薯未萎焉,野生型木薯明显萎焉,基因编辑木薯的SOD、POD活性和Pro含量均显著高于野生型木薯,MDA含量显著低于野生型,基因编辑木薯的叶片颜色更浅,胁迫响应基因SOD和WRKY31在基因编辑木薯中的表达量显著高于野生型;在低温胁迫后,基因编辑木薯和野生型木薯均萎焉,SOD活性、POD活性、MDA含量、叶片颜色和胁迫响应基因在基因编辑木薯与对照差异不显著。以上结果表明,基因编辑MeERF127提高了木薯的抗旱性和耐盐性,但对低温胁迫不响应,推测MeERF127可能调控SOD和WRKY31基因以响应干旱和盐胁迫。本研究结果为揭示MeERF127基因在木薯响应干旱和盐胁迫中的作用提供参考。

Drought,high salinity and low temperature severely damage plant cells,inhibit plant growth,and significantly reduce crop yields.Ethylene-responsive factors(ERFs)in the AP2/ERF superfamily play a key role in plant growth and development,and in stress responses.To investigate the role of MeERF127 in the response of cassava to abiotic stress,this study cloned the MeERF127 gene from cassava and conducted sequence alignment,subcellular localization,transcriptional activity analysis,and expression pattern analysis.A gene-edited vector for MeERF127 was constructed to obtain transgenic cassava,and the phenotype and physiological indicators after drought,salt and cold stress treatments were analyzed,along with the expression of stress-responsive genes.The results showed that the full-length CDS region of the MeERF127 gene was 711 bp,and the amino acid sequence contained YRG and RAYD elements at the N-and C-termini,respectively,with alanine and aspartic acid at positions 14 and 19,indicating that MeERF127 belonging to the ERF subfamily.MeERF127 was localized in the nucleus and had transcription factor activity.MeERF127 was most highly expressed in stems,with the lowest expression in callus tissues,and its expression peaks during the tuber formation stage(80 days after planting).Its expression level increased after drought and salt stress and slightly decreased after cold stress.A gene-edited vector was constructed,and Agrobacterium-mediated transformation of cassava callus tissues resulted in MeERF127 gene-edited lines with 22 and 3 base pair deletions,as confirmed by Hi-TOM high-throughput sequencing.After drought and salt stress treatments,the gene-edited cassava did not wilt,while the wild-type cassava showed significant wilting.The gene-edited cassava had significantly higher SOD and POD enzyme activities and Pro content,and significantly lower MDA content compared to the wild-type cassava.The leaf color of the gene-edited cassava was lighter,and the expression levels of stress-responsive genes SOD and WRKY31 were significantly higher in the gene-edited cassava than those in the wild-type.After cold stress,both gene-edited and wild-type cassava wilted,with no significant differences in SOD and POD enzyme activities,MDA content,leaf color and stress-responsive gene expression between the gene-edited and wild-type cassava.These results suggest that the gene-edited MeERF127 enhances the drought and salt tolerance of cassava but does not respond to cold stress,indicating that MeERF127 may regulate the SOD and WRKY31 genes to respond to drought and salt stress.The findings of this study would provide insights into the role of the MeERF127 gene in cassava's response to drought and salt stress.