甘蓝型油菜BnNF-YA1的克隆和功能鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2021-0959

摘要/Abstract

摘要：

NF-YA是组成NF-Y转录因子的3个亚基之一,在植物中参与生长发育、胁迫响应以及与微生物互作等重要生物过程。通过克隆BnNF-YA1,并对BnNF-YA1进行生物信息学分析和表达模式分析,分离克隆其编码序列,构建过表达载体,转化甘蓝型油菜,通过阳性鉴定获得BnNF-YA1的转基因阳性植株,并对过表达BnNF-YA1的转基因植株进行了逆境表型鉴定。结果表明,BnNF-YA1的开放读码框为858 bp,编码285个氨基酸;烟草瞬时表达分析发现BnNF-YA1蛋白定位于细胞核内;酵母生化分析表明BnNF-YA1全长蛋白没有转录激活活性;表达分析结果显示,BnNF-YA1受PEG处理诱导表达,且在幼叶、花苞和受精后20 d的角果中表达水平较高;过表达BnNF-YA1的甘蓝型油菜对PEG和MV处理的耐受性提高,表明BnNF-YA1可能参与调控甘蓝型油菜对渗透胁迫和氧化胁迫的响应。

关键词: 油菜, 转录因子, NF-YA, 过表达, 非生物逆境胁迫

Abstract:

NF-YA is one of the three subunits of NF-Y（nuclear factor Y）transcription factors,which is involved in plant growth and development,stress response,and interaction with microorganisms in plants. The bioinformatics and expression pattern analysis of cloned BnNF-YA1 were carried out. The coding sequence of the BnNF-YA1 was isolated and cloned. The overexpression vector of the BnNF-YA1 was constructed and transformed into rapeseed. The positive transgenic plants were identified,and the performances of the transgenic plants overexpressing BnNF-YA1 under stress treatments were evaluated. The results showed that the length of open reading frame of BnNF-YA1 was 858 bp,encoding 285 amino acids. Tobacco-transient expression analysis uncovered that BnNF-YA1 protein was localized in the nucleus. Biochemical analysis using yeast system showed that there was no transactivation activity in BnNF-YA1 full-length protein. Expression analysis revealed that the transcript abundance of BnNF-YA1 was induced by PEG treatment,and BnNF-YA1 showed a relatively high expression in the seedling leaf,bud,and 20 DAP silique. The overexpression of BnNF-YA1 increased the tolerance of transgenic rapeseed plants to PEG and methyl viologen（MV）treatments,suggesting that BnNF-YA1 may be involved in regulating the responses of rapeseed to osmotic and oxidative stress.

Key words: Brassica napus L., transcription factor, NF-YA, overexpression, abiotic stress

林科运, 段钰晶, 王高升, 孙念礼, 方玉洁, 王幼平. 甘蓝型油菜BnNF-YA1的克隆和功能鉴定[J]. 生物技术通报, 2022, 38(4): 106-116.

LIN Ke-yun, DUAN Yu-jing, WANG Gao-sheng, SUN Nian-li, FANG Yu-jie, WANG You-ping. Cloning and Functional Identification of BnNF-YA1 in Brassica napus L.[J]. Biotechnology Bulletin, 2022, 38(4): 106-116.

图/表 8

图1 BnNF-YA1亲/疏水性、磷酸化位点、二级结构、三级结构分析 A：BnNF-YA1亲/疏水性分布图;B：BnNF-YA1磷酸化位点预测;C：BnNF-YA1二级结构预测;D：BnNF-YA1三级结构预测

Fig. 1 Analysis of hydrophilicity/hydrophobicity,phos-phorylation site,secondary structure and tertiary structure of BnNF-YA1 A：Hydrophilic/hydrophobic analysis of BnNF-YA1. B：Prediction of phosphorylation sites of BnNF-YA1. C：Prediction of secondary structure of BnNF-YA1. D：Prediction of the tertiary structure of BnNF-YA1

图2 BnNF-YA1同源序列比对 Bn：油菜B. napus L;Bo：甘蓝B. oleracea;Br：白菜B. rapa;At：拟南芥Arabidopsis thaliana;Os：水稻Oryza sativa;Gm：大豆Glycine max;Zm：玉米Zea mays

Fig. 2 Multiple sequence alignments of BnNF-YA1 and its homologs

图3 NF-YA1系统发生分析 Zm：玉米Zea mays;Ata：山羊草Aegilops tauschii;Gm：大豆Glycine max;Os：水稻Oryza sativa;Sb：高粱Sorghum bicolor;Pt：白毛杨Populus tomentosa;Sl：番茄Solanum lycopersicum;St：马铃薯S. tuberosum;Bn：甘蓝型油菜Brassica napus L.;Bo：甘蓝B. oleracea;Br：白菜B. rapa;Rs：萝卜Raphanus sativus;At：拟南芥Arabidopsis thaliana;Gh：陆地棉Gossypium hirsutum;Me：木薯Manihot esculenta;Ah：花生Arachis hypogaea

Fig. 3 Phylogenetic analysis of NF-YA1 proteins

图4 BnNF-YA1表达模式分析 A：BnNF-YA1在聚乙二醇（PEG）、低温（cold）、高温（heat）以及脱落酸（ABA）处理条件下的表达变化情况;B：BnNF-YA1在甘蓝型油菜不同组织器官中的表达情况

Fig. 4 Expression profile of BnNF-YA1 A：Changes of BnNF-YA1 expression in B. napus under PEG,cold,heat,and ABA treatments. B：Spatial-temporal expression profile of BnNF-YA1 in the different organs of B. napus

图5 BnNF-YA1-OE过表达材料获得 A：过表达骨架载体示意图;B：BnNF-YA1 T0代过表达植株的PCR阳性鉴定;C：BnNF-YA1-OE转基因阳性植株RNA抽提检测;D：BnNF-YA1-OE转基因阳性植株反转录效果检测;E：BnNF-YA1-OE T0代过表达植株表达量鉴定

Fig. 5 Generation of BnNF-YA1-OE transgenic rapeseed plants A：Schematic diagram of the overexpresed vector backbone. B：PCR positive identification of overexpresed BnNF-YA1 in T0 generation. C：RNA detection of BnNF-YA1-OE transgenic positive plants. D：Reverse transcription detection of BnNF-YA1-OE transgenic positive plants. E：Expression of BnNF-YA1-OE in the T0 generation of overexpressed plants

图6 BnNF-YA1-OE T2代转基因植株子叶期表型鉴定 A：BnNF-YA1-OE T2代转基因植株子叶期表型;B：BnNF-YA1-OE T2代转基因植株下胚轴、根长及鲜重统计。误差表示标准差,*表示差异显著（P<0.05）,**表示差异极显著（P<0.01）

Fig. 6 Phenotypic identification of T2 generation of BnNF-YA1-OE transgenic plants at the cotyledon stage A：Phenotype of T2 generation of BnNF-YA1-OE transgenic plants at the cotyledon stage. B：The hypocotyl length,root length and fresh weight of T2 generation of BnNF-YA1-OE transgenic plants. The error bars represent the standard deviation（SD）. * represents significant difference（P<0.05）,and ** represents extremely significant difference（P<0.01）

图7 BnNF-YA1蛋白亚细胞定位

Fig. 7 Subcellular localization of BnNF-YA1 protein

图8 BnNF-YA1全长蛋白自激活活性分析

Fig. 8 Transactivation activity analysis of BnNF-YA1 full-length protein

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