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作物学报 ›› 2023, Vol. 49 ›› Issue (2): 402-413.doi: 10.3724/SP.J.1006.2023.24031

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

钙调素基因(HcCaM7)及其蛋白乙酰化修饰参与红麻响应非生物胁迫的作用

黄震1(), 吴启境1, 陈灿妮1, 吴霞1, 曹珊1, 张辉1, 岳娇1, 胡亚丽1, 罗登杰1, 李赟1, 廖长君3, 李茹2, 陈鹏1,*()   

  1. 1广西大学农学院 / 广西高校植物遗传育种重点实验室, 广西南宁 530004
    2广西大学生命科学与技术学院, 广西南宁 530004
    3广西博世科环保科技股份有限公司, 广西南宁 530007
  • 收稿日期:2022-01-25 接受日期:2022-06-07 出版日期:2022-07-04 网络出版日期:2022-07-04
  • 通讯作者: 陈鹏
  • 作者简介:E-mail: 549935163@qq.com
  • 基金资助:
    国家自然科学基金项目(31960368);财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-16-E14);广西博世科环保科技股份有限公司合作项目(GXU-BFY-2020-015)

Role of calmodulin gene (HcCaM7) and its protein acetylation is involved in kenaf response to abiotic stress

HUANG Zhen1(), WU Qi-Jing1, CHEN Can-Ni1, WU Xia1, CAO Shan1, ZHANG Hui1, YUE Jiao1, HU Ya-Li1, LUO Deng-Jie1, LI Yun1, LIAO Chang-Jun3, LI Ru2, CHEN Peng1,*()   

  1. 1College of Agriculture, Guangxi University / Key Laboratory of Plant Genetics and Breeding, Nanning 530004, Guangxi, China
    2College of Life Science and Technology, Guangxi University, Nanning 530004, Guangxi, China
    3Guangxi Bossco Environmental Protection Technology, Nanning 530007, Guangxi, China
  • Received:2022-01-25 Accepted:2022-06-07 Published:2022-07-04 Published online:2022-07-04
  • Contact: CHEN Peng
  • Supported by:
    National Natural Science Foundation of China(31960368);China Agriculture Research System of MOF and MARA(CARS-16-E14);Guangxi Bossco Environmental Protection Technology Co., Ltd.(GXU-BFY-2020-015)

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摘要：

钙调素是一类钙依赖性调节蛋白, 参与植物的生长发育、抗逆胁迫等多种生物学过程。本课题组前期通过蛋白质乙酰化修饰组学研究发现, 红麻钙调素蛋白7的乙酰化修饰参与了红麻花粉的发育调控。为研究其参与抗逆性的机制, 本研究以红麻保持系P3B双核期的花药为材料, 使用PCR法克隆了钙调素基因HcCaM7, 最大开放阅读框(open reading frame, ORF)为450 bp, 其由149个氨基酸组成, 编码相对分子质量16.85 kD的蛋白; 亚细胞定位结果显示, HcCaM7蛋白的表达主要定位在细胞质和细胞膜中; 利用病毒诱导的基因沉默技术沉默HcCaM7基因, 导致红麻沉默植株的生长受到抑制; 进一步在体外采用基因密码子扩展技术对发生乙酰化修饰氨基酸位点进行突变, 成功获得具有体外乙酰化修饰位点的蛋白HcCaM7mut, 并成功诱导表达了无乙酰化修饰的蛋白HcCaM7, 结果表明HcCaM7蛋白发生乙酰化修饰后可以显著促进NADK (NAD激酶)活性; 用点板法检测含有HcCaM7蛋白和HcCaM7mut蛋白的重组菌在盐(400 mmol L-1和500 mmol L-1 NaCl)、干旱(400 mmol L-1和600 mmol L-1甘露醇)、重金属(30 mmol L-1和50 μmol L-1 CdCl2)及低温胁迫后(利用液氮反复冻融模拟)在LB固体培养基上的存活率发现, 含有HcCaM7蛋白的重组菌存活率显著高于空载对照菌, 而含有乙酰化修饰的HcCaM7mut蛋白的重组菌存活率进一步提升, 表明HcCaM7蛋白能够提高大肠杆菌对非生物胁迫的耐受性, 并且乙酰化修饰后效果更佳。因此, HcCaM7基因可以调控红麻生长发育和响应非生物胁迫, 乙酰化修饰可以促进HcCaM7蛋白发挥作用。

关键词: 红麻, 钙调素蛋白7, 蛋白乙酰化修饰, 病毒诱导的基因沉默, 基因密码子扩展技术, 非生物胁迫

Abstract:

Calmodulin (CaM) is a kind of calcium-dependent regulatory proteins involved in plant growth and development, stress tolerance, and other biological processes. In the previous study in kenaf acetylome, our team found that the protein acetylation modification calmodulin protein7 (CaM7) was involved in the regulation pollen development in kenaf. In order to explore its specific mechanism, we cloned the calmodulin gene HcCaM7 from kenaf P3B binuclear anther by using the PCR cloning way. Its maximum open reading frame (ORF) was 450 bp, encoding a protein containing 149 amino acids with a molecular weight of 16.85 kD. Subcellular localization revealed that HcCaM7 was mainly located in cytoplasm and cell membrane. Silencing HcCaM7 by virus induced gene silencing technique caused growth inhibition in kenaf. Furthermore, the protein HcCaM7mut with acetylation modification site was successfully obtained in vitro and the expression of HcCaM7 without acetylation modification was successfully induced. Acetylation of HcCaM7 protein significantly promoted NADK (NAD kinase) activity, indicating HcCaM7 acetylation involved in its functional regulation. The recombinant bacteria containing HcCaM7 protein and HcCaM7mut protein were detected in NaCl (400 mmol L-1 and 500 mmol L-1 NaCl), drought (400 mmol L-1 and 600 mmol L-1 mannitol) and heavy metals (30 μmol L-1 and 50 μmol L-1) by dot plate method. The results showed that the survival rate of recombinant bacteria containing HcCaM7 protein was significantly higher than that of empty control bacteria, in addition, the survival rate of recombinant bacteria containing acetylated HcCaM7mut protein was further improved. The results indicated that HcCaM7 protein could enhance the abiotic stress resistance of E.coli, and the effect of acetylation modification was better. Therefore, HcCaM7 gene regulated the growth and development of kenaf and abiotic stress resistance, and acetylation modification could promote the role of HcCaM7 protein.

Key words: kenaf, calmodulin protein 7, protein acetylation modification, virus induced gene silencing, gene codon extension technology, abiotic stress

表1

研究所用引物列表"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
HcCaM7-F ATGGCCGATCAGCTCACC
HcCaM7-R CTTGGCCATCATCACTTTGA
HcCaM7-GFP-F CAGGATATCCAGATCCAGTGGGATCCATGGCCGATCAGCTCACC
HcCaM7-GFP-R TAAGCTTGGTACCGAGCTCACCCGGGGATCCCTTGGCCATCATCACTTTGA
HcCaM7- pTRV2-F GATTCTGTGAGTAAGGTTACCGAATTCGGCGGAACTCCAAGATATGA
HcCaM7- pTRV2-R CCCCATGGAGGCCTTCTAGAGAATTCACCATCAACATCGGCTTCAC
HcCaM7mut-F GGTGTTTGACtAGGATCAGAATGGTTTCATATCTGC
HcCaM7mut-R GATCCTaGTCAAACACCCTGAATGCCTCTTTA
HcCaM7-pET32α-F AAGCTTGTCGACGGAGCTC GAATTCCTTGGCCATCATCACTTTGA
HcCaM7-pET32α-R CATGGCTGATATCGGATCC GAATTCATGGCCGATCAGCTCACC
HcCaM7-qPCR-F GATGCTGATGGAAACGGG
HcCaM7-qPCR-R CCATCACCATCAACATCGG

图1

HcCaM7基因的克隆及表达分析 A: HcCaM7的PCR扩增产物; M: DL2000 marker; 1: HcCaM7 ORF扩增。B: HcCaM7基因在红麻P3A和P3B不同组织中表达分析。数据为3个生物学重复±标准差。柱上不同小写字母表示差异显著(P < 0.05)。"

图2

HcCaM7的生物信息学分析 A: HcCaM7蛋白保守结构域的预测; B: HcCaM7蛋白三级结构预测结果; C: HcCaM7蛋白的系统发育树分析。"

图3

HcCaM7蛋白亚细胞定位 A: Mcherry通道; B: GFP通道; C: 明场; D: 融合场。"

图4

HcCaM7基因VIGS沉默分析 A: 转基因植株的qRT-PCR分析, pTRV2空载为对照, 1~9代表HcCaM7基因沉默植株; B: 沉默HcCaM7植株的表型分析; C: CaM7-pTRV2红麻植株鲜重; D: CaM7-pTRV2红麻植株株高。数据为3个生物学重复±标准差; 不同小写字母表示之间差异显著(P < 0.05)。"

图5

红麻P3A和P3B花药中HcCaM7基因和其蛋白在表达水平定量分析 A: HcCaM7在红麻P3A和P3B花药中相对表达量; B: HcCaM7蛋白在红麻P3A和P3B花药中表达水平, 1: P3A的花药, 2: P3B的花药。数据为3个生物学重复±标准差; 不同小写字母表示之间差异显著(P < 0.05)。"

图6

HcCaM7蛋白乙酰化修饰位点定点突变"

图7

HcCaM7蛋白及其点突变蛋白纯化结果及His标签和乙酰化修饰检测 A: HcCaM7mut蛋白纯化结果, 1、2、3为纯化结果; B: HcCaM7蛋白纯化结果, 1、2、3为纯化结果; C: His-Tag Western-blot检测结果, 1是HcCaM7mut蛋白, 2是HcCaM7蛋白; D: 蛋白乙酰化修饰Western blot检测结果, 1是HcCaM7mut蛋白, 2是HcCaM7蛋白。"

图8

HcCaM7乙酰化修饰对NADK酶活的影响 数据为3个生物学重复±标准差。不同小写字母表示之间差异显著(P < 0.05)。"

图9

HcCaM7在大肠杆菌中应对非生物胁迫的抗性检测 A: 盐胁迫; B: 甘露醇胁迫; C: CdCl2胁迫; D: 冷胁迫; a, b, c, d: 胁迫后稀释了104倍的菌落存活数。数据为3个生物学重复±标准差。不同小写字母表示之间差异显著(P < 0.05)。"

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