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作物学报 ›› 2018, Vol. 44 ›› Issue (8): 1159-1168.doi: 10.3724/SP.J.1006.2018.01159

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆半胱氨酸蛋白酶抑制剂基因GmCYS2的功能鉴定

柯丹霞(),彭昆鹏,贾妍,曾硕,王英枝,张静怡   

  1. 信阳师范学院生命科学学院 / 大别山农业生物资源保护与利用研究院, 河南信阳 464000
  • 收稿日期:2017-11-23 接受日期:2018-03-26 出版日期:2018-08-10 网络出版日期:2018-04-20
  • 通讯作者: 柯丹霞
  • 基金资助:
    国家自然科学基金项目(31400213);河南省科技攻关计划项目(182102110448);信阳师范学院青年骨干教师资助计划项目(2015);信阳师范学院“南湖学者奖励计划”青年项目(2016)

Functional Characterization of Soybean Cystatins Gene GmCYS2

Dan-Xia KE(),Kun-Peng PENG,Yan JIA,Shuo ZENG,Ying-Zhi WANG,Jing-Yi ZHANG   

  1. College of Life Sciences / Institute for Conservation and Utilization of Agro-bioresources in Dabie Mountains, Xinyang Normal University, Xinyang, Henan 464000, China
  • Received:2017-11-23 Accepted:2018-03-26 Published:2018-08-10 Published online:2018-04-20
  • Contact: Dan-Xia KE
  • Supported by:
    the National Natural Science Foundation of China(31400213);Henan Province Science and Technology Research Projects(182102110448);the Funding Scheme for Young Core Teachers of Xinyang Normal University(2015);Nanhu Scholars Program for Young Scholars of XYNU(2016)

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摘要：

半胱氨酸蛋白酶抑制剂(cystatin, CYS)在结瘤, 根瘤发育和衰老过程中起重要作用。本研究克隆了1个大豆CYS家族基因GmCYS2, 氨基酸序列比对及进化树分析表明, GmCYS2与木豆(Cajanus cajan) CYS相似性最高。在体外对该基因编码蛋白进行了表达和纯化, 重组蛋白GmCYS2的酶活性抑制实验显示, 该蛋白对L类和B类组织蛋白酶的抑制活性明显高于对H类组织蛋白酶, 并且在30 d根瘤提取物中的抑制活性高于在60 d根瘤提取物中。此外, 构建GmCYS2的植物表达载体, 通过百脉根毛根转化法获得转GmCYS2基因的超表达复合体植株。GmCYS2的超量表达增加了百脉根的结瘤数目, 并且上调共生标记基因的表达。结果说明GmCYS2蛋白具有一定的酶活性抑制作用, 并且正调控百脉根共生结瘤过程。

关键词: 大豆, 半胱氨酸蛋白酶抑制剂, 半胱氨酸蛋白酶, 共生结瘤, 百脉根

Abstract:

CYS (cystatin) plays an important role in nodulation, nodule development and senescence. In this study, we cloned a soybean CYS family gene GmCYS2, which had the highest similarity with pigeonpea (Cajanus cajan) CYS, shown by amino acid sequence alignment and phylogenetic tree analysis. The gene encoding protein was expressed and purified in vitro. Enzyme activity inhibition experiment of recombinant protein GmCYS2 showed that the cathepsin inhibitory activity of GmCYS2 protein on L and B classes was higher than that on H class, and cathepsin inhibitory activity in nodule extract from nodule at 30 d was generally higher than that at the 60 d. In addition, the plant overexpression vector was constructed and the GmCYS2-overexpression composite plants were obtained by Lotus japonicus hairy root transformation method. Overexpression of GmCYS2 increased the number of nodules in Lotus japonicus plants and up regulated the expression levels of symbiotic marker genes. These results suggest that GmCYS2 protein can inhibit the enzyme activity and positively regulate the symbiotic nodulation of Lotus japonicus.

Key words: Glycine max, cystatin, cysteine protease, symbiotic nodulation, Lotus japonicus

表1

本研究中所使用的引物"

引物名称
Primer name		 引物序列
Sequence of primer (5′-3′)
F-GmCYS2-pro. TACGGGGGATTGGTC
R-GmCYS2-pro. TCACTGCGTGGAAGGAG
F-OX CGggatccATGGCGGCGTTGATAAG
R-OX GGggtaccTCACTGCGTGGAAGGAG
F-NIN-rt AACTCACTGGAAACAGGTGCTTTC
R-NIN-rt CTATTGCGGAATGTATTAGCTAGA
F-ENOD40-1-rt GGAGGTATGCTCAAACATTC
R-ENOD40-1-rt GTAACTTCTCAAGAGAAGACC
F-ENOD40-2-rt CAAAACTCGTTATGTTGCGG
R-ENOD40-2-rt CACCTCAAAGGAAGAAGAACA
F-GmCYS2-rt CAACAAGTGGTGTCTG
R-GmCYS2-rt TCACTGCGTGGAAGGAG
F-Polyubiquitin TTCACCTTGTGCTCCGTCTTC
R-Polyubiquitin AACAACAGCACACACAGACAATC

图1

大豆GmCYS2与其他植物中同源蛋白的序列比对(A)及进化树分析(B) 图A中用黄色、红色、绿色和蓝色方框标出半胱氨酸蛋白酶抑制剂的主要保守结构域。图B中标尺代表遗传相似性, 指不同植物间同源蛋白进化关系的远近。"

图2

His-GST-GmCYS2融合蛋白的序列分析 氨基酸第5~10位: His标签; 第14~233位: GST标签; 第244~250位: TEV(烟草蚀纹病毒)切割位点; 251~355位: GmCYS2蛋白序列(去掉N端26个氨基酸编码的信号肽)。"

图3

GmCYS2蛋白的体外表达、抗体检测和蛋白纯化 A: SDS-PAGE分析蛋白在BL21(DE3)菌株中的表达情况; B: Western blot分析, 抗-His抗体检测; C: 纯化蛋白的SDS-PAGE分析。M1: 蛋白marker; M2: Western marker; M3: 蛋白预染marker; PC1: BSA (1 μg); PC2: BSA (2 μg); NC: 未诱导的全细胞裂解液。1: 15℃诱导16 h的全细胞裂解液; 2: 37℃诱导4 h的全细胞裂解液; NC1: 未诱导的细胞裂解液上清液; NC2: 未诱导的细胞裂解液沉淀; 3: 15℃诱导16 h的细胞裂解液上清液; 4: 15℃诱导16 h的细胞裂解液沉淀; 5: 37℃诱导4 h的细胞裂解液上清液; 6: 37℃诱导4 h的细胞裂解液沉淀; 7: 从37℃诱导4 h的细胞裂解液上清液中纯化的蛋白。"

图4

重组蛋白GmCYS2在不同时期根瘤提取物中的抑制活性分析 样品分别为30 d和60 d根瘤提取物。反应液中加入纯化的GmCYS2蛋白, 另外分别加入组织蛋白酶L类、B类和H类3种不同底物, 试验重复3次。* P < 0.05。"

图5

GmCYS2基因的过量表达对百脉根共生结瘤的影响 A: 复合体植株接种MAFF303099根瘤菌30 d后的结瘤表型。毛状根表达pMUb:GmCYS2 (GmCYS2-OX)或空载体pU1301 (CK), Bar = 10 mm; B: 单株复合体百脉根植株的平均结瘤数目; C: 根据单株根瘤数划分的不同结瘤种类所占的相对百分比; D: 阳性毛根的GUS鉴定; E: RT-PCR检测阳性毛根中GmCYS2基因的表达。M: DL2000 DNA marker; 1: 阳性质粒对照; 2: 空载体对照; 3~5: 复合体植株。"

表2

过表达GmCYS2复合体百脉根植株的结瘤数目统计"

试验
Experiment		 对照植株
CK		 超表达植株
GmCYS2-OX		 P值
P-value
1 5.42±1.53 (n=30) 10.33±2.35 (n=25) < 0.01
2 6.18±2.05 (n=23) 9.87±2.48 (n=20) < 0.01

图6

转基因毛根中GmCYS2以及共生基因的转录水平检测 荧光定量PCR检测GmCYS2以及共生基因 NIN、ENOD40-1和ENOD40-2 在超表达植株(GmCYS2-OX)和对照植株(CK)阳性毛根中的转录水平。* P<0.05, ** P<0.01。"

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