梭梭NAC转录因子HaNAC38功能及特性分析

doi:10.13304/j.nykjdb.2022.0266

摘要/Abstract

摘要：

从梭梭干旱转录组中获得与干旱相关的NAC转录因子基因HaNAC38，为了研究其功能，以过表达HaNAC38基因拟南芥纯合株系作为对象，比较转基因株系与野生型间的差异，并利用酵母单杂交技术对HaNAC38转录因子可能结合的DNA核心序列进行验证。结果表明，在自然干旱和模拟盐胁迫条件下，过表达HaNAC38基因拟南芥株系生长状况显著优于野生型拟南芥。过表达HaNAC38基因拟南芥株系的种子发芽率、幼苗叶片脯氨酸含量及过氧化氢酶活性均显著高于野生型拟南芥株系，丙二醛含量和过氧化氢含量显著低于野生型拟南芥。由此表明HaNAC38显著增强了拟南芥对于干旱和盐胁迫的耐受能力。酵母单杂交试验结果表明，HaNAC38转录因子可以在酵母体内特异性结合TTGCGT核心序列，并激活下游报告基因的表达。以上结果为明确梭梭HaNAC38转录因子的功能及其调控网络奠定了基础。

关键词: 梭梭, NAC, 转录因子, 功能分析, 结合特性

Abstract:

In order to study the function of HaNAC38， a drought-related NAC transcription factor obtained from Haloxylon ammodendron drought transcriptomes， the homozygous lines of Arabidopsis thaliana overexpressing HaNAC38 genewere used as the materials， and the differences of growth and physiological indices were compared between plant of overexpressing HaNAC38 gene and wild type. The binding activity of HaNAC38 transcription factor and DNA core sequence was verified by yeast single hybridization technique. The results showed， under natural drought and simulated salt stress， the growth of Arabidopsis thaliana overexpressing HaNAC38 gene was significantly better than that of the wild type. The seed germination rate and the proline content and catalase activity in leaf of plant overexpressing HaNAC38 gene were significantly higher than those of wild type， while the contents of malondialdehyde and hydrogen peroxide were significantly lower than those of wild type. It showed that HaNAC38 significantly enhanced the tolerance of Arabidopsis thaliana to drought and salt stress. The yeast single hybridization indicated that the DNA core sequence of HaNAC38 transcription factor could bind TTGCGT core sequence specifically in yeast and activate the expression of downstream reporter gene. The above results laid a foundation for clarifying the function and regulatory network of haloxylon ammodendrite HaNAC38 transcription factor.

Key words: Haloxylon ammodendron, NAC transcription factor, functional analysis, binding characteristics

中图分类号:

Q786

罗广科, 穆榕博, 薛冰, 张桦, 任燕萍, 麻浩. 梭梭NAC转录因子HaNAC38功能及特性分析[J]. 中国农业科技导报, 2023, 25(10): 65-73.

Guangke LUO, Rongbo MU, Bing XUE, Hua ZHANG, Yanping REN, Hao MA. Function and Characteristic Analysis of Haloxylon ammodendron NAC Transcription Factor HaNAC38[J]. Journal of Agricultural Science and Technology, 2023, 25(10): 65-73.

图/表 8

表1 实验所用引物序列

Table 1 Sequences of primers used in experiment

引物名称 Primer name	引物序列 Primer sequence（5’-3’）
HaNAC38-F	ATGGAGAGTGGTAAGATGACCGTT
HaNAC38-R	TTAAAGTTTTTTTCTAATGCTATTGCAG
PPT-F	ATGAGCCCAGAACGACGCC
PPT-R	TCAAATCTCGGTGACGGGCA
HaNAC38BDL-F	AGGTCTTTGCTTGTCCTTTGCC
HaNAC38BDL-R	CTTCCCAGGCTTGGTGTCAC
Actin-F	TTCCTCATGCCATCCTCCGTCTT
Actin-R	CAGCGATACCTGAGAACATAGTGG
HaNAC38-pGADT7-F	GTACCAGATTACGCTCATATGATGGAGAGTGGTAAGATGACCGTT
HaNAC38-pGADT7-R	ACGATTCATCTGCAGCTCGAGTTAAAGTTTTTTTCTAATGCTATTGCAG
Core-pHis2-F	CGGAATTCTTTTTGCGTTTGTTGCGTTTTTTGCGTTGAGCTCG
Core-pHis2-R	CGAGCTCAACGCAAAAAACGCAACAAACGCAAAAAGAATTCCG
mCore-pHis2-F	CGGAATTCTTTTTGAGTTTGTTGAGTTTTTTGAGTTGAGCTCG
mCore-pHis2-R	CGAGCTCAACTCAAAAAACTCAACAAACTCAAAAAGAATTCCG

图1 HaNAC38转基因拟南芥及野生型拟南芥发芽率A：盐胁迫；B：干旱胁迫； *和**分别表示转基因株系与野生型间在P<0.05和P<0.01水平差异显著

Fig. 1 Germination rates of HaNAC38 transgenic plant and wild type of Arabidopsis thalianaA：Salt stress； B：Drought stress； * and ** indicate significant differences between transgenic plant and wild type at P<0.05 and P<0.01 levels， respectively

图2 干旱胁迫条件下拟南芥各株系生长状况

Fig. 2 Growth status of Arabidopsis thaliana under drought stress

图3 干旱胁迫条件下拟南芥叶片生理指标注：不同小写字母表示同一处理下不同株系间在P<0.05水平差异显著。

Fig. 3 Physiological indices of Arabidopsis thaliana leaves under drought stressNote： Different lowercase letters indicate significant differences between different strains under same treatment at P<0.05 level.

图4 拟南芥叶片离体失水率注：**表示转基因株系与野生型株系间在P<0.01水平差异显著。

Fig. 4 Ex vivo water loss rate of Arabidopsis thaliana leavesNote：** indicates significant difference between transgenic plants and wild type at P<0.01 level.

图5 盐胁迫条件下拟南芥各株系生长状况

Fig. 5 Growth status of Arabidopsis thaliana under salt stress

图6 盐胁迫条件下拟南芥叶片生理指标注：不同小写字母表示同一处理下不同株系间在P<0.05水平差异显著。

Fig. 6 Physiological indices of Arabidopsis thaliana leaves under salt stressNote： Different lowercase letters indicate significant differences between different plants under same treatment at P<0.05 level.

图7 HaNAC38与DNA核心序列在酵母体内的结合A：HaNAC38与TTGCGT串联的结合验证；B：HaNAC38与TTGAGT串联的结合验证

Fig. 7 Binding of HaNAC38 to the DNA core sequence in yeastA： Combined verification between HaNAC38 and TTGCGT in series； B： Combination verification between HaNAC38 and TTGAGT in series

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