雪莲SikCDPK1启动子的克隆和活性分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0570

摘要/Abstract

摘要：

旨在克隆雪莲（Saussurea involucrata）SikCDPK1 启动子并分析其活性,为进一步解析雪莲 SikCDPK1基因的转录调控机制奠定基础。通过TAIL-PCR技术从雪莲中克隆SikCDPK1启动子,利用PlantCARE分析启动子区顺式作用元件,构建全长启动子或5'端缺失启动子驱动的GUS重组表达载体 P0∷GUS、P1∷GUS、P2∷GUS和P3∷GUS,转入根癌农杆菌中进行瞬时转化试验,通过GUS组织化学染色分析不同长度启动子的活性,分别测定低温和干旱胁迫下的GUS酶活。结果显示,获得了1 042 bp的SikCDPK1启动子序列,pSikCDPK1具有真核生物启动子核心元件TATA-box和CAAT-box,还含有多个与逆境、激素、光响应等相关的顺式作用元件。转化的烟草叶片经GUS染色之后均显蓝色,启动活性依次为P0>P1>P2>P3,低温、干旱处理后GUS酶活性发生变化。SikCDPK1启动子被成功克隆,具有驱动下游报告基因表达的活性。

关键词: 雪莲, SikCDPK1启动子, GUS活性, 瞬时表达

Abstract:

The objective is to clone the SikCDPK1 promoter of Saussurea involucrata and analyze its activity, so as to lay the foundation for further understanding the transcriptional regulation mechanism of the SikCDPK1 gene in S. involucrata. TAIL-PCR was used to clone the promoter of SikCDPK1 from S. involucrata, and PlantCARE to analyze the promoter cis-acting elements. The recombinant expression vector P0∷GUS, P1∷GUS, P2∷GUS and P3∷GUS driven by full-length promoter or 5'-deletion promoter were constructed and transferred into Agrobacterium tumefaciens for transient transformation. The activities of different length promoters were analyzed by GUS staining, and the activity of GUS under low temperature and drought was determined respectively. As results, a 1 042 bp promoter sequence of SikCDPK1 was obtained. pSikCDPK1 had the core elements of eukaryotic promoter TATA-box and CAAT-box, and also contained several cis-acting elements related to stress, hormone and light response. All transformed tobacco leaves showed blue color after GUS staining, and the activity was in P0>P1>P2>P3. The GUS activity changed after low temperature and drought. In sum, the promoter of SikCDPK1 is successfully cloned and has the activity of driving downstream reporter gene expression.

Key words: Saussurea involucrata, SikCDPK1 promoter, GUS activity, transient expression

史光珍, 王兆晔, 孙琦, 朱新霞. 雪莲SikCDPK1启动子的克隆和活性分析[J]. 生物技术通报, 2022, 38(9): 191-197.

SHI Guang-zhen, WANG Zhao-ye, SUN Qi, ZHU Xin-xia. Cloning and Activity Analysis of SikCDPK1 Promoter from Saussurea involucrata[J]. Biotechnology Bulletin, 2022, 38(9): 191-197.

图/表 7

表1 本研究所用引物

Table 1 Primers used in this study

引物Primer name	引物序列Primer sequence（5'-3'）	用途Purpose
AD1	NTCGASTWTSGWGTT	TAIL-PCR
AD2	NGTCGASWGANAWGAAAA	TAIL-PCR
AD3	WGTCNACWANCANACA	TAIL-PCR
AD4	TGWGNAGWANCANAGA	TAIL-PCR
AD5	AGWGNAGWANCAWAGG	TAIL-PCR
SP1	CGTTATCCCAAACTGCCCTTGTCCTA	TAIL-PCR
SP2	TCCGTTAGGGGTAGGTGGGGTATCTT	TAIL-PCR
SP3	TTCGGTCCAACACAAGTATTCCCCAT	TAIL-PCR
pSikCDPK1-F1	CCCAAGCTTCCTTAGCATCTATGAGGGTCG	启动子克隆 Promoter cloning
pSikCDPK1-R1	GACTAGTTCCAACACAAGTATTCCCCATG	启动子克隆 Promoter cloning
pSikCDPK1-F2	CCCAAGCTTCTCTTCTTATGGGTTCAAAGGGTCA	5'端缺失分析 5'-end deletion analysis
pSikCDPK1-F3	CCCAAGCTTAAGCGTCATGCCAGTCAAGC	5'端缺失分析 5'-end deletion analysis
pSikCDPK1-F4	CCCAAGCTTTTTCAATGGAGAAGCGACGAGC	5'端缺失分析 5'-end deletion analysis

图1 SikCDPK1启动子驱动的GUS表达载体

Fig. 1 GUS expression vectors driven by SikCDPK1 promoters

图2 SikCDPK1基因启动子的克隆 A：第一轮PCR扩增;B：第二轮PCR扩增;C：第三轮PCR扩增。M：DNA2000 marker;1：AD1;2：AD2;3：AD3;4：AD4

Fig. 2 Cloning of SikCDPK1 promoter A：The first round of PCR amplification. B：The second round of PCR amplification. C：The third round of PCR amplification. M：DNA2000 marker. 1：AD1. 2：AD2. 3：AD3. 4：AD4

表2 启动子 pSikCDPK1序列中的顺式作用元件及功能

Table 2 Cis-elements and functions in the promoter pSikCDPK1 sequence

顺式作用元件名称 Name of cis-acting element	序列 Sequence（5'-3'）	功能 Function	位置 Location/ nt
O2-site	GATGA（C/T）（A/G）TG（A/G）或GATGATGTGG	玉米醇溶蛋白代谢调节 Zein metabolism regulation	-56 to -48,-596 to -587
CGTCA-motif	CGTCA	茉莉酸甲酯响应 MeJA-responsiveness	-94 to -89,-562 to -557,-479 to -474
CBFHV	RYCGAC	低温反应元件 Cis-acting element for cold	-103 to -97,-379 to -373,-672 to -666
GT1GMSCAM4	GAAAAA	盐诱导响应相关元件 NaCl-induced	-159 to -153
LTRECOREATCOR15	CCGAC	冷诱导低温反应元件 Cis-acting element for cold induction	-215 to -210,-459 to -454
GC-motif	GCCCCC	参与缺氧特异性诱导的类增强子Enhancer-like element involved in anoxic specific inducibility	-287 to -281
TGACG-motif	TGACG	茉莉酸甲酯响应 MeJA-responsiveness	-310 to -305,-499 to -494
ABRE	TACGGTC	ABA响应Abscisic acid responsiveness	-327 to -320
W-box	TTGACC	WRKY转录因子的结合位点WRKY transcription factor binding site	-387 to -381
box-S	AGCCACC	无功能No function	-571 to -564
MYC	CAATTG	无功能No function	-533 to -539
G-box	CACGTC	光响应 Light responsiveness	-689 to -683
MYB	CAACCA	MYB 顺式作用元件MYB Cis-acting element	-759 to -753
OSE2ROOTNODULE	CTCTT	在根瘤的感染细胞中被激活 Activated in infected cells of root nodules	-821 to -816
MYB-Core	CGTTAG	脱水胁迫反应元件Responsive to dehydration	-958 to -952
DPBFCOREDCDC3	ACACNNG	脱落酸响应元件ABA-responsive elements	-975 to -967
ACGTATERD1	ACGT	脱水诱导反应元件 Early responsive to dehydration	-1 038 to -1 034

图3 SikCDPK1启动子的PCR扩增及表达载体双酶切鉴定 A：PCR扩增SikCDPK1启动子（M：DNA2000 marker;1,2：P0的PCR产物;3,4：P1的PCR产物;5,6：P2的PCR产物;7,8：P3的PCR产物）;B,C：表达载体双酶切鉴定（B1：P0的双酶切;B2：P0的质粒;B3：P1的双酶切;B4：P1的质粒;C1：P2的双酶切;C2：P2的质粒;C3：P3的双酶切;C4：P3的质粒;M：DNA marker III）

Fig. 3 PCR amplification of promoter SikCDPK1 and double restriction identification of expression vector A：PCR amplification SikCDPK1 promoter（M：DNA2000 marker. 1,2：PCR product of P0. 3,4：PCR product of P1. 5,6：PCR product of P2. 7,8：PCR product of P3）. B,C：Double digestion identification of expression vector（B1：P0 double digestion. B2：P0 plasmid. B3：P1 double digestion. B4：P1 plasmid. C1：P2 double digestion. C2：P2 plasmid. C3：P3 double digestion. C4：P3 plasmid. M：DNA marker III）

图4 SikCDPK1启动子瞬时转化烟草GUS染色分析 A：阴性对照（非转基因型）;B：阳性对照（35S∷GUS）;C：P0;D：P1;E：P2;F：P3

Fig. 4 GUS staining analysis of transient transformed tobacco with SikCDPK1 promoter A：Negative control（Non-transgenic）. B：Positive control（35S∷GUS）. C：P0. D：P1. E：P2. F：P3

图5 胁迫处理下GUS染色分析及酶活力的测定 A：低温胁迫后的 GUS 染色和酶活力情况;B：PEG胁迫后的 GUS 染色和酶活力情况。a：p35S-GUS-1304阳性对照;b：pSikCDPK1-GUS-1304实验组;c：常温下实验组对照

Fig. 5 Gus staining analysis and enzyme activity determin-ation under stress treatment A：Gus staining and enzyme activity after low temperature stress. B：Gus staining and enzyme activity after PEG stress. a：Positive control p35S-GUS-1304. b：Experimental group pSikCDPK1-GUS-1304. c：Experimental group control at room temperature

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