盐碱胁迫诱导的猴樟酵母cDNA文库构建及CbP5CS上游调控因子筛选

doi:10.13560/j.cnki.biotech.bull.1985.2023-0022

摘要/Abstract

摘要：

盐碱土壤环境是限制猴樟引种推广的主要因素，脯氨酸是植物适应盐碱胁迫过程中主要的渗透调节物质，筛选并鉴定出调控脯氨酸合成的转录因子对于猴樟耐盐碱分子机理研究具有重要意义。以猴樟实生苗为材料，在水培培养基础上，采用0 mmol/L和10 mmol/L的Na₂CO₃溶液分别进行处理，选取处理6 h、48 h的根系组织，提取总RNA，构建盐碱胁迫诱导的猴樟根系酵母cDNA文库；以猴樟根系总DNA为模板，克隆CbP5CS启动子序列，采用Y1H酵母单杂交技术筛选出与CbP5CS启动子存在互作的蛋白，并通过高通量测序技术鉴定出调控猴樟脯氨酸合成的转录因子。结果表明，所构建的cDNA文库库容为4.88×10⁷CFU/mL，总克隆数为9.76×10⁷ CFU，文库插入片段平均长度在1 000 bp左右，cDNA片段重组率为100%，符合酵母杂交试验的要求。克隆出的CbP5CS启动子序列长2 012 bp，成功构建出诱饵质粒pHIS2-CbP5CS，利用共转化方法从文库中筛选到31个与CbP5CS互作的EST序列。经酵母回转验证，31个EST序列均与CbP5CS存在互作。经NGS测序和BLAST比对搜索，获得6个转录因子基因，分别为锌指CCCH结构域蛋白25异构体 x1、AtbHLH104、转录因子 TFIIIC、RING/FYVE/PHD锌指超家族蛋白、GATA型锌指转录因子家族蛋白、AtbHLH96。以上结果为进一步研究植物脯氨酸代谢响应盐碱胁迫的分子机制奠定基础。

关键词: 盐碱胁迫, 酵母cDNA文库, CbP5CS, 猴樟, 上游调控因子, 转录因子

Abstract:

The saline-alkali soil is a major factor limiting the introduction and popularization of Cinnamomun bodinieri. Proline is one of the most important compatible solutes that plants accumulate for osmotic adjustment in response to saline-alkali stress. Therefore, it is of great significance to identify the key transcription factors that regulate the biosynthesis of proline and illustrate the underlying molecular mechanisms. In the present study, the seedlings were treated with 0 and 10 mmol/L Na₂CO₃ solution on the basis of hydroponic culture, respectively. Total RNA was extracted from the root tissues at 6 and 48 h after treatment to construct the yeast cDNA library induced by saline-alkali stress. The promoter sequence of CbP5CS was cloned using total DNA of C. bodinieri root system as template. The titer of the library was 4.88×10⁷CFU/mL, and the total clone number was 9.76×10⁷ CFU. The average size of the inserts was 1 000 bp, and the recombination efficiency was 100%, which met the requirements of yeast hybridization test. The length of the cloned promoter of CbP5CS was 2 012 bp, and the decoy plasmid pHIS2-CbP5CS was constructed successfully. Then the promoter region of CbP5CS was cloned and the CbP5CS promoter interacting proteins was screened by yeast one hybrid and high-throughput sequencing. The 31 unique CbP5CS-interatcting ESTs were identified from the library by co-transformation, 32 EST sequences were interacted with CbP5CS verified via yeast rotation. Six of them were annotated as transcription factors, including GW020491（zinc finger CCCH domain-containing protein 25 isoform X1）, GW028183（AtbHLH104）, GW000650（transcription factor TFIIIC）, GW007525（RING/FYVE/PHD zinc finger superfamily protein）, GW015686（GATA type zinc finger transcription factor family protein）, GW027120（AtbHLH96）. These results provide a basis for further study on the molecular mechanism of plant proline metabolism in response to saline-alkali stress.

Key words: saline-alkali stress, yeast cDNA library, CbP5CS, Cinnamomun bodinieri, upstream regulators, transcription factor

韩浩章, 张丽华, 李素华, 赵荣, 王芳, 王晓立. 盐碱胁迫诱导的猴樟酵母cDNA文库构建及CbP5CS上游调控因子筛选[J]. 生物技术通报, 2023, 39(9): 236-245.

HAN Hao-zhang, ZHANG Li-hua, LI Su-hua, ZHAO Rong, WANG Fang, WANG Xiao-li. Construction of cDNA Library of Cinnamomun bodinieri Induced by Saline-alkali Stress and Screening of CbP5CS Upstream Regulators[J]. Biotechnology Bulletin, 2023, 39(9): 236-245.

图/表 12

参考文献 26

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引物名称Primer name	序列Sequence（5'-3'）	应用Application
CDS III/3' PCR primer	AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG	逆转录
SMART IV oligonucleotide	ATTCTAGAGGCCGAGGCGGCCGACATGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN	逆转录
P1-F	CATATGGCCATGGAGGCCAGTGAATTCAAGCAGTGGTATCAACGCAGAGTGG	ds cDNA扩增
P2-F	CATATGGCCATGGAGGCCAGTGAATTCAAAGCAGTGGTATCAACGCAGAGTGG	ds cDNA扩增
P3-F	CATATGGCCATGGAGGCCAGTGAATTCAAAAGCAGTGGTATCAACGCAGAGTGG	ds cDNA扩增
P4-R	CATCTGCAGCTCGAGCTCGATGGATCCCTAGAGGCCGAGGCGGCCGACATG	ds cDNA扩增
Prime M1	AAGCAGTGGTATCAACGCAGAGT	均一化
T7-F	taatacgactcactatagggcgag	菌落PCR
AD-R	GCACGATGCACAGTTGAAG	菌落PCR

引物名称Primer name	序列Sequence（5'-3'）	应用Application
CDS III/3' PCR primer	AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG	逆转录
SMART IV oligonucleotide	ATTCTAGAGGCCGAGGCGGCCGACATGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN	逆转录
P1-F	CATATGGCCATGGAGGCCAGTGAATTCAAGCAGTGGTATCAACGCAGAGTGG	ds cDNA扩增
P2-F	CATATGGCCATGGAGGCCAGTGAATTCAAAGCAGTGGTATCAACGCAGAGTGG	ds cDNA扩增
P3-F	CATATGGCCATGGAGGCCAGTGAATTCAAAAGCAGTGGTATCAACGCAGAGTGG	ds cDNA扩增
P4-R	CATCTGCAGCTCGAGCTCGATGGATCCCTAGAGGCCGAGGCGGCCGACATG	ds cDNA扩增
Prime M1	AAGCAGTGGTATCAACGCAGAGT	均一化
T7-F	taatacgactcactatagggcgag	菌落PCR
AD-R	GCACGATGCACAGTTGAAG	菌落PCR

位点 Site	位置 Position	序列 Sequence	重复次数 Number of repetitions	功能 Function
ABRE	-1 713	ACGTG	1	脱落酸响应元件
AE-box	+191	AGAAACAA	1	部分光响应模块
ARE	+680、-1 262、-693	AAACCA	3	厌氧诱导必需元件
AT~TATA-box	+551	TATATA	1	光响应元件
Box 4	-1 846	ATTAAT	1	光响应中的保守DNA模块
CAAT-box	+58、+75、-99、+139、+184、+196、+414、+513、+704、-743、-753、+770、+817、-819、+869、-902、+914、+941、+942、+1 005、-1 008、+1 115、 -1 118、1 140、-1 168、+1 195、+1 198、-1 275、 -1 293、+1 348、-1 351、-1 394、-1 429、+1 479、 +1 480、-1 498、+1 523、+1 540、+1 541、+1 570、+1 591、+1 617、-1 636、-1 813	CAAAT/CAAT/CCAAT/TGCCAAC	44	启动子和增强子区域普通顺式作用元件
CAT-box	+1 976	GCCACT	1	分生组织表达响应元件
CGTCA-motif	-143、-732、+146	CGTCA	3	茉莉酸甲酯响应的元件
ERE	-1 743	ATTTCATA	1	乙烯响应元件
G-Box	+1 713	CACGTT	1	光响应元件
G-box	+80	CACGAC	1	光响应元件
GT1-motif	-610、+1 225、+665	GGTTAA/GTGTGTGAA	3	光响应元件
MYB	+2、-1 258、+1 201、-1 927、+1 001、-1 224	CAACAG/CAACCA/TAACCA/	6	MYB转录因子结合位点
MYB-like sequence	-1 224、-1 927	TAACCA	2	MYB转录因子结合位点
MYC	-139、+817、-184	CATTTG/CAATTG	3	bHLH转录因子结合位点
Myb	-1 154、+1 485	TAACTG	2	MYB转录因子结合位点
Myb-binding site	+2、+1 201	CAACAG	2	MYB转录因子结合位点
P-box	-158	CCTTTTG	1	赤霉素响应元件
SARE	-992	TTCGACCATCTT	1	水杨酸响应元件
STRE	-265、+1 080、+1 801、+572、+1 962、-1 402、 -1 834、-281、-712	AGGGG	9	压力响应元件
TATA-box	+202、-550、+267、+551、+552、+553、-1 161、 -1 162、+1 178、-1 179、-1 180、+1 471、-1 472、 -1 473、+1 554、-1 741、+1 850、-1 851、-1 917	TATA/TATATAA/ccTATAAAaa/TATATA/ATATAA/ATATAT/ATTATA/TATAA/TACAAAA	19	-30位置附近核心启动子转录起始位点
TATC-box	-258	TATCCCA	1	赤霉素响应元件
TCA-element	-989、+1 408	CCATCTTTTT	2	水杨酸响应元件
TCCC-motif	+1 870	TCTCCCT	1	光响应元件
TGACG-motif	+143、+732、-146	TGACG	3	茉莉酸甲酯响应元件
Unnamed	-234、+1 640、+256、-1 712	CGTGG	4	未知
Unnamed	-628	TCCACGTAGA	1	未知
Unnamed	-9、-1 083、+643、+1 871、-568、1 377、-833、 -1 191、-536、+1 863、+710、-635、-921	CTCC	13	未知
WRE3	-829、+1 865、-1 098	CCACCT	3	愈伤调节响应元件
WUN-motif	-559	AAATTACT	1	愈伤调节响应元件

位点 Site	位置 Position	序列 Sequence	重复次数 Number of repetitions	功能 Function
ABRE	-1 713	ACGTG	1	脱落酸响应元件
AE-box	+191	AGAAACAA	1	部分光响应模块
ARE	+680、-1 262、-693	AAACCA	3	厌氧诱导必需元件
AT~TATA-box	+551	TATATA	1	光响应元件
Box 4	-1 846	ATTAAT	1	光响应中的保守DNA模块
CAAT-box	+58、+75、-99、+139、+184、+196、+414、+513、+704、-743、-753、+770、+817、-819、+869、-902、+914、+941、+942、+1 005、-1 008、+1 115、 -1 118、1 140、-1 168、+1 195、+1 198、-1 275、 -1 293、+1 348、-1 351、-1 394、-1 429、+1 479、 +1 480、-1 498、+1 523、+1 540、+1 541、+1 570、+1 591、+1 617、-1 636、-1 813	CAAAT/CAAT/CCAAT/TGCCAAC	44	启动子和增强子区域普通顺式作用元件
CAT-box	+1 976	GCCACT	1	分生组织表达响应元件
CGTCA-motif	-143、-732、+146	CGTCA	3	茉莉酸甲酯响应的元件
ERE	-1 743	ATTTCATA	1	乙烯响应元件
G-Box	+1 713	CACGTT	1	光响应元件
G-box	+80	CACGAC	1	光响应元件
GT1-motif	-610、+1 225、+665	GGTTAA/GTGTGTGAA	3	光响应元件
MYB	+2、-1 258、+1 201、-1 927、+1 001、-1 224	CAACAG/CAACCA/TAACCA/	6	MYB转录因子结合位点
MYB-like sequence	-1 224、-1 927	TAACCA	2	MYB转录因子结合位点
MYC	-139、+817、-184	CATTTG/CAATTG	3	bHLH转录因子结合位点
Myb	-1 154、+1 485	TAACTG	2	MYB转录因子结合位点
Myb-binding site	+2、+1 201	CAACAG	2	MYB转录因子结合位点
P-box	-158	CCTTTTG	1	赤霉素响应元件
SARE	-992	TTCGACCATCTT	1	水杨酸响应元件
STRE	-265、+1 080、+1 801、+572、+1 962、-1 402、 -1 834、-281、-712	AGGGG	9	压力响应元件
TATA-box	+202、-550、+267、+551、+552、+553、-1 161、 -1 162、+1 178、-1 179、-1 180、+1 471、-1 472、 -1 473、+1 554、-1 741、+1 850、-1 851、-1 917	TATA/TATATAA/ccTATAAAaa/TATATA/ATATAA/ATATAT/ATTATA/TATAA/TACAAAA	19	-30位置附近核心启动子转录起始位点
TATC-box	-258	TATCCCA	1	赤霉素响应元件
TCA-element	-989、+1 408	CCATCTTTTT	2	水杨酸响应元件
TCCC-motif	+1 870	TCTCCCT	1	光响应元件
TGACG-motif	+143、+732、-146	TGACG	3	茉莉酸甲酯响应元件
Unnamed	-234、+1 640、+256、-1 712	CGTGG	4	未知
Unnamed	-628	TCCACGTAGA	1	未知
Unnamed	-9、-1 083、+643、+1 871、-568、1 377、-833、 -1 191、-536、+1 863、+710、-635、-921	CTCC	13	未知
WRE3	-829、+1 865、-1 098	CCACCT	3	愈伤调节响应元件
WUN-motif	-559	AAATTACT	1	愈伤调节响应元件

基因ID Gene ID	读数 Read count	拟南芥同源序列 Arabidopsis thaliana homolog	E值E-value	功能注释 Functional annotation
GW020491	1	AT3G47120.1	8.00E-76	锌指 CCCH结构域蛋白25异构体 x1
GW028183	15	AT4G14410.2	6.00E-51	AtbHLH104
GW000650	3	AT3G15420.1	1.00E-24	转录因子 TFIIIC
GW009089	7	AT4G00755.2	6.00E-59	F-box 家族蛋白
GW009287	8	AT1G64230.2	3.00E-83	泛素连接酶
GW012884	24	AT5G15730.2	2.00E-137	蛋白激酶超家族
GW000705	24	AT4G02340.1	2.00E-47	α/β 水解酶超家族蛋白
GW003120	4	AT5G04480.1	0	葡萄糖基转移酶超家族蛋白
GW004800	7	AT5G63030.1	4.00E-42	硫氧还蛋白超家族蛋白
GW006791	16	ATMG01360.1	0	细胞色素氧化酶
GW007525	13	AT3G47550.6	6.00E-41	RING/FYVE/PHD锌指超家族蛋白
GW008185	15	AT2G20360.1	1.00E-161	NAD（P）结合罗斯曼卷曲超家族蛋白
GW008711	19	AT4G14305.1	8.00E-79	过氧化物酶体膜22 kD（Mpv17/PMP22）家族蛋白
GW010514	3	AT2G27030.3	2.00E-82	钙调素
GW011043	24	AT4G35450.1	3.00E-115	锚蛋白重复序列含有蛋白
GW011868	16	AT4G26220.1	3.00E-80	S-腺苷甲硫氨酸依赖的甲基转移酶超家族蛋白
GW011935	13	AT1G55160.1	2.00E-35	功能未知的蛋白质
GW012148	1	AT1G54290.1	3.00E-48	翻译起始因子eIF1
GW012186	19	AT5G42850.2	4.00E-40	硫氧还蛋白超家族蛋白
GW014171	56	AT2G44860.2	2.00E-62	核糖体蛋白质 L24e 家族蛋白
GW014289	34	AT2G27020.1	1.00E-129	20s 蛋白酶体α亚基G1
GW014358	24	AT1G68590.1	1.00E-43	核糖体蛋白质 PSRP-3/Ycf65
GW014549	10	AT4G32140.1	4.00E-98	类EamA转运家族蛋白
GW015686	1	AT1G10200.1	2.00E-80	GATA型锌指转录因子家族蛋白
GW015796	64	AT1G13950.1	4.00E-80	真核延伸因子5A-1
GW017908	15	AT3G55440.1	4.00E-111	磷酸丙糖异构酶
GW020994	23	AT5G53560.1	2.00E-48	细胞色素 B5亚型E
GW022429	11	ATMG01360.1	0	细胞色素氧化酶
GW024339	14	AT4G19420.1	2.00E-153	果胶乙酰酯酶家族蛋白
GW025798	12	AT1G53680.1	1.00E-69	谷胱甘肽S-转移酶TAU28
GW027120	11	AT1G72210.1	4.00E-52	AtbHLH96