生物技术通报 ›› 2023, Vol. 39 ›› Issue (11): 308-317.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0096
张玉娟1(), 黎冬华2, 宫慧慧1, 崔新晓1, 高春华1, 张秀荣1, 游均2(), 赵军胜1()
收稿日期:
2023-02-08
出版日期:
2023-11-26
发布日期:
2023-12-20
通讯作者:
赵军胜,男,博士,研究员,研究方向:经济作物遗传育种;E-mail: zhaojunshengsd@163.com;作者简介:
张玉娟,女,博士,助理研究员,研究方向:植物逆境分子生物学;E-mail: 723949246@qq.com
基金资助:
ZHANG Yu-juan1(), LI Dong-hua2, GONG Hui-hui1, CUI Xin-xiao1, GAO Chun-hua1, ZHANG Xiu-rong1, YOU Jun2(), ZHAO Jun-sheng1()
Received:
2023-02-08
Published:
2023-11-26
Online:
2023-12-20
摘要:
NAC转录因子在植物生长发育及盐胁迫响应过程中具有重要的调控作用。芝麻SiNAC77参与盐胁迫响应过程。克隆芝麻SiNAC77并分析其耐盐功能,为芝麻耐盐育种提供分子基础和基因资源。克隆SiNAC77的全长CDS序列,利用生物信息学软件对其基因序列和氨基酸序列特征进行分析。构建SiNAC77过表达载体,利用农杆菌介导法转化拟南芥。对过表达株系的耐盐表型和生理生化指标进行分析。结果表明,成功克隆了长度为1 008 bp的SiNAC77 CDS序列,编码335个氨基酸,相对分子量为135.93 kD,预测等电点为4.91,含有33个潜在的磷酸化位点;启动子区域含有MBS、STRE、ARE、ABRE和TCA等多个非生物胁迫及激素响应元件。成功构建了SiNAC77过表达载体,并转化拟南芥,获得12个独立的转基因阳性株系。在盐胁迫下,与野生型拟南芥相比,过表达株系的种子发芽率、初生根长度、鲜重均显著提高,转基因株系中Na+/K+和相对MDA含量显著降低,相对SOD和POD抗氧化酶活性则显著增强。过表达芝麻SiNAC77可以增强抗氧化酶活性,减少离子毒害和氧化损伤,提高转基因植株的耐盐性。
张玉娟, 黎冬华, 宫慧慧, 崔新晓, 高春华, 张秀荣, 游均, 赵军胜. 芝麻NAC转录因子基因SiNAC77的克隆及耐盐功能分析[J]. 生物技术通报, 2023, 39(11): 308-317.
ZHANG Yu-juan, LI Dong-hua, GONG Hui-hui, CUI Xin-xiao, GAO Chun-hua, ZHANG Xiu-rong, YOU Jun, ZHAO Jun-sheng. Cloning and Salt-tolerance Analysis of NAC Transcription Factor SiNAC77 from Sesamum indicum L.[J]. Biotechnology Bulletin, 2023, 39(11): 308-317.
引物名称Primer name | 引物序列Primer sequence(5'-3') | 用途Purpose |
---|---|---|
NAC77-BamH I-F | tgtctagactcgagggatccATGGAACAGTTGGCCGGTTTTG | 基因克隆Gene clone |
NAC77-BamH I-R | agcctgcagccatggggatccTCAGTAGTTCCACAAGCAGTC | |
35s-F | GACGCACAATCCCACTATCC | 转基因植株检测Transgenic plant detection |
NAC77-R | TCAGTAGTTCCACAAGCAGTC | |
Actin-F | CGGCGAGTCATCGTCATCAATGT | 拟南芥内参引物Internal primer of Arabidopsis thaliana |
Actin-R | GTTCGGGATTACGGTGGTGGGA | |
NAC77-Fq | CTCCACTCACGGACTTGTCA | 荧光定量PCR引物Fluorescent quantitative PCR primer |
NAC77-Rq | AGGAAAGGAGGGTTGGATTG |
表1 本研究所用引物序列
Table 1 Primer sequence used in this study
引物名称Primer name | 引物序列Primer sequence(5'-3') | 用途Purpose |
---|---|---|
NAC77-BamH I-F | tgtctagactcgagggatccATGGAACAGTTGGCCGGTTTTG | 基因克隆Gene clone |
NAC77-BamH I-R | agcctgcagccatggggatccTCAGTAGTTCCACAAGCAGTC | |
35s-F | GACGCACAATCCCACTATCC | 转基因植株检测Transgenic plant detection |
NAC77-R | TCAGTAGTTCCACAAGCAGTC | |
Actin-F | CGGCGAGTCATCGTCATCAATGT | 拟南芥内参引物Internal primer of Arabidopsis thaliana |
Actin-R | GTTCGGGATTACGGTGGTGGGA | |
NAC77-Fq | CTCCACTCACGGACTTGTCA | 荧光定量PCR引物Fluorescent quantitative PCR primer |
NAC77-Rq | AGGAAAGGAGGGTTGGATTG |
图4 转基因拟南芥植株的鉴定 A:卡那霉素筛选T1转基因苗;B:T1转基因植株的PCR鉴定,1-12为转基因植株,“-”为纯水空白对照,“+”为质粒对照,WT为野生型植株对照,M为DL2000 marker;C:T2转基因拟南芥中SiNAC77的表达量检测,OE-1、OE-2和OE-3分别来自T1“4、5、8”转基因拟南芥家系。*表示在P<0.05水平上差异显著,**表示在P<0.01水平上差异显著。下同
Fig. 4 Identification of transgenic Arabidopsis plants A: Identification of T1 transgenic Arabidopsis plants by kanamycin. B: Identification of T1 transgenic Arabidopsis plants, 1-12 indicate transgenic lines, “-” indicates pure water blank control, “+” indicates plasmid control, WT indicates wild type of Arabidopsis plants control, and M indicates DL2000 marker. C: Expression of SiNAC77in transgenic Arabidopsis. OE-1, OE-2, and OE-3 were obtained from T1(4, 5 and 8)transgenic plants. * indicate significant difference at P<0.05 level; **indicates significant difference at P<0.01 level. The same below
图5 转基因拟南芥的耐盐性鉴定 A:盐胁迫对转基因拟南芥种子发芽率的影响;B:盐胁迫对转基因拟南芥初生根根长的影响;C:盐胁迫对转基因拟南芥生物积累量的影响;D:盐胁迫下野生型和转基因拟南芥植株的表型
Fig. 5 Resistance characteristics of transgenic Arabidopsis plants to salt stress A: Seed germination of transgenic Arabidopsis under salt stress. B: The elongation of the primary root of transgenic Arabidopsis under salt stress. C: Fresh weight of transgenic Arabidopsis under salt stress. D: Phenotypes of wild type and transgenic Arabidopsis plants under salt stress
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