异子蓬SabHLH169基因的克隆及抗旱功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0049

摘要/Abstract

摘要：

碱性螺旋-环-螺旋（basic belix-loop-helix，bHLH）转录因子是真核生物中最大的转录因子家族之一，其成员广泛参与植物生长、发育和胁迫响应。前期在异子蓬（Suaeda aralocaspica）光合作用关键酶PEPC-1的研究中筛选到一个可能与SaPEPC-1启动子相互作用的bHLH转录因子SabHLH169，探明该基因功能将为后期异子蓬bHLH基因的深入研究奠定基础。通过克隆获得该基因，采用生物信息学、实时荧光定量PCR等方法，初步探究SabHLH169基因的耐旱功能。结果显示，SabHLH169包含2 100 bp核苷酸，编码699个氨基酸，其蛋白质C末端具有典型的bHLH结构域，原核表达和Western blot分析表明，SabHLH169重组蛋白能够正确表达，其分子量大小与预测结果一致。过表达SabHLH169的拟南芥转基因株系在萌发期具有更高的耐旱性；干旱胁迫下，转基因拟南芥中3个干旱胁迫相关基因AtRD22、AtRD29A和AtDREB2A以及3个光合作用关键酶基因AtLhcb2、AtRubicso和AtPEPC的表达水平显著升高，且AtPEPC酶活性被显著增强。由此推测，SabHLH169基因可能在干旱胁迫响应中具有潜在功能。

关键词: 异子蓬, bHLH, 干旱胁迫, 转基因拟南芥, 光合关键酶

Abstract:

The basic helix-loop-helix（bHLH）transcription factor（TF）is one of the largest TF families in eukaryotes, its members are widely involved in plant growth, development and response to stress. In our previous work of studying photosynthesis key enzyme PEPC-1 in Suaeda aralocaspica, a bHLH TF（SabHLH169）potentially interacting with the SaPEPC-1 promoter was screened. In the present study, the biological characteristics and preliminary function of this gene was explored, which may provide evidence for further studies of bHLH related genes in S. aralocaspica. The gene was obtained by homogous cloning, and the drought-resistant properties of SabHLH169 gene were characterized by bioinformatics and qRT-PCR analyses. The results showed that the open reading frame（ORF）of SabHLH169 contained 2100 bp nucleotides, encoding an protein containing 699 amino acids. The protein had a typical bHLH structural domain at the C-terminus. The prokaryotic expression and Western blot of the recombinant protein showed that the protein was expressed correctly, and the size was consistent with the predicted result. Arabidopsis thaliana transgenic lines of SabHLH169 presented higher drought tolerance at germination stage. Under drought stress, three drought-responsive genes（AtRD22, AtRD29A and AtDREB2A）and three photosynthesis key enzyme genes（AtLhcb2, AtRubicso and AtPEPC）were up-regulated in transgenic A. thaliana. Meanwhile, the PEPC enzyme activity was significantly enhanced. It is speculated that SabHLH169 gene may be involved in response to drought stress.

Key words: Suaeda aralocaspica, bHLH, drought stress, transgenic A. thaliana, photosynthesis key enzyme

鄢梦雨, 韦晓薇, 曹婧, 兰海燕. 异子蓬SabHLH169基因的克隆及抗旱功能分析[J]. 生物技术通报, 2023, 39(11): 328-339.

YAN Meng-yu, WEI Xiao-wei, CAO Jing, LAN Hai-yan. Cloning of Basic Helix-loop-helix（bHLH）Transcription Factor Gene SabHLH169 in Suaeda aralocaspica and Analysis of Its Resistances to Drought Stress[J]. Biotechnology Bulletin, 2023, 39(11): 328-339.

图/表 10

表1 本文所用引物序列

Table 1 Primer sequences used in this study

名称 Name	上游引物序列 Forward primer（5'-3'）	下游引物序列 Reverse primer（5'-3'）
SabHLH169（PCR）	ATGGCTACTCACTTGCAGCAGTTGCTTC	TCAGGCATTAGTTTTTGATTGAAGTAGTTGG
pSuper1300-SabHLH169	GCGTCGACATGGCTACTCACTTGCAGCAGTTGCT	CGGGATCCTCAGGCATTAGTTTTTGATTGAAGTA
pMal-c2X-SabHLH169	CGGGATCCATGGCTACTCACTTGCAGCAGTTGCTTC	GCGTCGACTCAGGCATTAGTTTTTGATTGAAGTAGTT
AtHPT	GGTCGCGGAGGCTATGGATGC	GCTTCTGCGGGCGATTTGTGT
AtRD22（qRT-PCR）	GACTTTCGATTTTACCGACGAG	CGCTACCGGTTTTACCTTTATG
AtDREB2A（qRT-PCR）	CGTTTCAGGATGAGATGTGTGA	CTCATCGTGCATATAAAACGCA
AtRD29A（qRT-PCR）	TTCTGTAAGGACGACGTTTACA	CGTACTCGTTACATCCTCTGTT
AtACTIN（qRT-PCR）	GGTAACATTGTGCTCAGTGGTGG	AACGACCTTAATCTTCATGCTGC
AtPEPC（qRT-PCR）	AGCCTTCAGGGAACCACAAT	CTCCAAAGACGGGTCGCATG
AtRubisco（qRT-PCR）	TGGCTTCCTCTATGCTCTCTTC	ACACTTGAGCGGAGTCGGTGCA
AtLhcb2（qRT-PCR）	ATGTTGGGTGCTCTCGGATG	CGCGTGGATCAAGTTAGGGT
AtRCA（qRT-PCR）	GGCCGCCGCAGTTTCCACCG	AAAGTTGTAGACACAGGTTCCA

图1 异子蓬总RNA的提取及SabHLH169 ORF的扩增 A：异子蓬幼苗总RNA.1, 2：两个总RNA样品；B：SabHLH169 ORF的PCR扩增. M1, M2：DL 2000, DL5000 DNA分子量标准

Fig. 1 Extraction of total RNA and PCR amplification of SabHLH169 ORF in S. aralocaspica A: Total RNA of S. aralocaspica seedlings. 1,2: Two samples of total RNA; B: PCR amplification of SabHLH169 ORF; M1, M2: DL2000, DL5000 DNA markers

图2 异子蓬SabHLH169氨基酸序列分析 N端下划线部分表示bHLH-MYC_N结构域；C端下划线部分表示HLH结构域

Fig. 2 Analysis of amino acid sequences of SabHLH169 in S. aralocaspica N-terminal underline indicates bHLH-MYC_N domain; C-terminal underline indicates HLH domain

图3 异子蓬SabHLH169系统进化树及HLH结构域分析 A：SabHLH169系统进化树分析. 系统发育树使用MEGA X软件基于NJ法构建，基于1 000次 bootstrap；Sa：Suaeda aralocaspica，异子蓬；Cq：Chenopodium quinoa，藜麦；Bv：Beta vulgaris，甜菜；At：Arabidopsis thaliana，拟南芥；Nt：Nicotiana tabacum，烟草；So：Spinacia oleracea，菠菜；Os：Oryza sativa，水稻；◆表示异子蓬中的bHLH169。B：SabHLH169与近缘物种的氨基酸序列多重比对。不同颜色背景表示保守程度不同（深蓝>粉红>浅蓝>灰色；黑色背景表示完全保守）；上划线表示HLH结构域

Fig. 3 Analysis of the phylogenetic tree and bHLH domain of SabHLH169 in S. aralocaspica A: Analysis of the phylogenetic tree of SabHLH169, which was constructed based on the NJ method using MEGA X software with 1 000 bootstraps. B: Multiple alignment of the bHLH motif of amino acid sequences between SabHLH169 and the closely related species. Different colored letters indicate different degrees of conservation（dark blue > pink red > light blue > grey; the dark background indicates sequence identical）; the upper line indicates the HLH domain

图4 异子蓬SabHLH169及其近缘种保守基序分析 A：排名前10的motif碱基富集示意图. 不同颜色方块代表motif1-motif10；字母高度代表该氨基酸出现的频率，高度越高频率越大；B：不同物种bHLH转录因子的保守motif分布. Sa：Suaeda aralocaspica，异子蓬；Cq：Chenopodium quinoa，藜麦；Bv：Beta vulgaris，甜菜；At：Arabidopsis thaliana，拟南芥；Nt：Nicotiana tabacum，烟草，So：Spinacia oleracea，菠菜；Os：Oryza sativa，水稻

Fig. 4 Analysis of the conserved motifs of SabHLH169 in C. quinoa and the closely related species A: Schematic diagram of the base enrichment of the top 10 motifs; boxes in different colors represent motif1-motif10; the height of the letter indicates the frequency of the amino acid, and the higher the height, the greater the frequency. B: Conserved motif analysis of bHLH transcription factors in different species

图5 原核表达载体pMal-c2X -SabHLH169的构建及重组蛋白SabHLH169的SDS-PAGE和Western blot分析 A：SabHLH169全长序列的扩增。B：pMal-c2X -SabHLH169的菌液PCR鉴定。-：阴性对照；C：重组质粒的双酶切鉴定。D：SDS-PAGE结果。M：蛋白质Marker；1：pMal-c2X-SabHLH169诱导前；2-4：pMal-c2X-SabHLH169诱导后（0.5、0.8、1.0 mmol/L IPTG）；5，6：分别为pMal-c2X诱导前和诱导后；E：Western blot 结果. 1：pMal-c2X -SabHLH169诱导后（0.5 mmol/L IPTG）；2：pMal-c2X-SabHLH169诱导前；3：pMal-c2X诱导前

Fig. 5 Construction of procaryotic expression vector pMal-c2X -SabHLH169 and SDS-PAGE and Western blot analysis of SabHLH169 A: Amplification of SabHLH169. B: PCR identification of pMal-c2X-SabHLH169 transformed colonies; -: negative control. C: Double digestion of recombinant plasmid. D: SDS-PAGE results. M: Protein marker; 1: Before induction of pMal-c2X-SabHLH169; 2-4: after induction of pMal-c2X-SabHLH169（0.5 mmol/L, 0.8 mmol/L, 1 mmol/L IPTG）; 5, 6: before and after induction of pMal-c2X. E: Western blot results; 1: After induction of pMal-c2X-SabHLH169（0.5 mmol/L IPTG）; 2: before induction of pMal-c2X-SabHLH169; 3: before induction of pMal-c2X

图6 植物表达载体pSuper1300-SabHLH169的构建及过表达SabHLH169转基因拟南芥株系的鉴定 A：SabHLH169的扩增；B：pSuper1300-SabHLH169重组菌液PCR鉴定；C：重组质粒的双酶切鉴定；D：过表达SabHLH169转基因拟南芥T0代的鉴定；E，F：3个T1转基因株系OE5、OE16、OE20中SabHLH169的PCR和RT-qPCR 鉴定. +：阳性对照；-：阴性对照；WT：野生型拟南芥；OE5、OE16和OE20：过表达 SabHLH169的3个拟南芥株系；M：DL 5000 DNA marker

Fig. 6 Construction of plant expression vector pSuper1300-SabHLH169 and identification of transgenic A. thaliana with SabHLH169 overexpression A: Amplification of SabHLH169. B: PCR identification of pSuper1300-SabHLH169 transformed colonies. C: Double digestion of recombinant plasmid. D: Identification of the T0 generation of transgenic A. thaliana overexpress SabHLH169. E, F: PCR and RT-qPCR identification of SabHLH169 in three T1 transgenic lines OE5, OE16, OE20. +: positive control; -: negative control. WT: Wild type A. thaliana; OE5, OE16 and OE20: three transgenic A. thaliana lines with SabHLH169 overexpression. M: DL 5000 DNA marker

表2 SabHLH169 转基因拟南芥T1代进行抗生素筛选3∶1分离比株系

Table 2 Antibiotic screening of SabHLH169 transgenic A. thaliana T1 generation for 3∶1 segregation ratio lines

株系 Strain	阳性苗 Positive seedlings	阴性苗 Negative seedlings	卡方检验 Chi-square test（P-value）	株系 Strain	阳性苗 Positive seedling	阴性苗 Negative seedling	卡方检验 Chi-square test（P-value）
OE1	134	37	0.310	OE13	78	31	0.407
OE5	81	26	0.867	OE14	64	27	0.304
OE6	70	33	0.099	OE16	65	26	0.431
OE7	79	40	0.030	OE17	62	26	0.325
OE8	78	32	0.322	OE18	54	25	0.173
OE11	64	29	0.169	OE19	54	26	0.121
OE12	61	24	0.491	OE20	56	21	0.645

图7 甘露醇处理下SabHLH169转基因拟南芥种子萌发实验 A：表型分析；B: 种子萌发率统计

Fig. 7 Seed germination of SabHLH169 transgenic A. thaliana under mannitol treatment A: Phenotype analysis. B: Statistics of seed germination percentage

图8 转基因拟南芥干旱和光合作用相关基因的表达及PEPC酶活性分析 A-C：转基因拟南芥中干旱胁迫相关基因的表达分析；D-G：光合作用相关基因的表达分析；H：转基因拟南芥 PEPC 酶活性分析。不同小写字母表示相同条件不同株系之间的基因表达量差异显著（P<0.05）

Fig. 8 Analysis of expression patterns of drought and photosynthesis related genes and PEPC enzyme activity in transgenic A. thaliana A-C: Expressions of drought stress-related genes in transgenic A.thaliana. D-G: Expressions of photosynthesis related genes. H: PEPC enzyme activity in transgenic A. thaliana. Different lowercase letters indicate significant differences in gene expression level at the same condition between different lines（P<0.05）

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