Cloning and Activity Analysis of SikCDPK1 Promoter from Saussurea involucrata

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

Abstract

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

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.

Figures/Tables 7

References 22

[1]	Li J, Liu HL, Xia WW, et al. De novo transcriptome sequencing and the hypothetical cold response mode of Saussurea involucrata in extreme cold environments[J]. Int J Mol Sci, 2017, 18(6):1155. doi: 10.3390/ijms18061155 URL
[2]	Singh A, Sagar S, Biswas DK. Calcium dependent protein kinase, a versatile player in plant stress management and development[J]. Crit Rev Plant Sci, 2017, 36(5/6):336-352. doi: 10.1080/07352689.2018.1428438 URL
[3]	Smale ST, Kadonaga JT. The RNA polymerase II core promoter[J]. Annu Rev Biochem, 2003, 72:449-479. pmid: 12651739
[4]	田晓涵. 两种不同生境植物CDPK1基因的克隆及功能初探[D]. 石河子: 石河子大学, 2016.
	Tian XH. Cloning and functional analysis of CDPK1 genes in two different habitats plants[D]. Shihezi: Shihezi University, 2016
[5]	Liu YG, Chen YL. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J]. BioTechniques, 2007, 43(5):649-650, 652, 654 passim. doi: 10.2144/000112601 URL
[6]	Shi SJ, Li SG, Asim M, et al. The Arabidopsis calcium-dependent protein kinases(CDPKs)and their roles in plant growth regulation and abiotic stress responses[J]. Int J Mol Sci, 2018, 19(7):1900. doi: 10.3390/ijms19071900 URL
[7]	Danino YM, Even D, Ideses D, et al. The core promoter:at the heart of gene expression[J]. Biochim Biophys Acta, 2015, 1849(8):1116-1131.
[8]	李旭娟, 林秀琴, 字秋艳, 等. 甘蔗ScMOC1基因启动子的克隆与瞬时表达分析[J]. 植物遗传资源学报, 2019, 20(3):709-717.
	Li XJ, Lin XQ, Zi QY, et al. Cloning and transient expression analysis of ScMOC1 promoter in sugarcane[J]. J Plant Genet Resour, 2019, 20(3):709-717.
[9]	Dang FF, Wang YN, She JJ, et al. Overexpression of CaWRKY27, a subgroup IIe WRKY transcription factor of Capsicum annuum, positively regulates tobacco resistance to Ralstonia solanacearum infection[J]. Physiol Plant, 2014, 150(3):397-411. doi: 10.1111/ppl.12093 URL
[10]	Dang FF, Wang YN, Yu L, et al. CaWRKY40, a WRKY protein of pepper, plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection[J]. Plant Cell Environ, 2013, 36(4):757-774. doi: 10.1111/pce.12011 URL
[11]	Eulgem T. Dissecting the WRKY web of plant defense regulators[J]. PLoS Pathog, 2006, 2(11):e126. doi: 10.1371/journal.ppat.0020126 pmid: 17121464
[12]	Eulgem T, Somssich IE. Networks of WRKY transcription factors in defense signaling[J]. Curr Opin Plant Biol, 2007, 10(4):366-371. pmid: 17644023
[13]	Knoth C, Ringler J, Dangl JL, et al. Arabidopsis WRKY70 is required for full RPP4-mediated disease resistance and basal defense against Hyaloperonospora parasitica[J]. Mol Plant Microbe Interact, 2007, 20(2):120-128. doi: 10.1094/MPMI-20-2-0120 URL
[14]	Wang YN, Dang FF, Liu ZQ, et al. CaWRKY58, encoding a group I WRKY transcription factor of Capsicum annuum, negatively regulates resistance to Ralstonia solanacearum infection[J]. Mol Plant Pathol, 2013, 14(2):131-144. doi: 10.1111/j.1364-3703.2012.00836.x URL
[15]	Bahn SC, Bae MS, Park YB, et al. Molecular cloning and characterization of a novel low temperature-induced gene, blti2, from barley(Hordeum vulgare L.)[J]. Biochim Biophys Acta, 2001, 1522(2):134-137. pmid: 11750066
[16]	Chen A, Gusta LV, Brûlé-Babel A, et al. Varietal and chromosome 2H locus-specific frost tolerance in reproductive tissues of barley(Hordeum vulgare L. )detected using a frost simulation chamber[J]. Theor Appl Genet, 2009, 119(4):685-694. doi: 10.1007/s00122-009-1079-1 URL
[17]	Chen A, Reinheimer J, Brûlé-Babel A, et al. Genes and traits associated with chromosome 2H and 5H regions controlling sensitivity of reproductive tissues to frost in barley[J]. Theor Appl Genet, 2009, 118(8):1465-1476. doi: 10.1007/s00122-009-0995-4 pmid: 19277599
[18]	Dunn MA, Goddard NJ, Zhang L, et al. Low-temperature-responsive barley genes have different control mechanisms[J]. Plant Mol Biol, 1994, 24(6):879-888. pmid: 8204825
[19]	Ivashuta S, Naumkina M, Gau M, et al. Genotype-dependent transcriptional activation of novel repetitive elements during cold acclimation of alfalfa(Medicago sativa)[J]. Plant J, 2002, 31(5):615-627. pmid: 12207651
[20]	Conforte AJ, Guimarães-Dias F, Neves-Borges AC, et al. Isolation and characterization of a promoter responsive to salt, osmotic and dehydration stresses in soybean[J]. Genet Mol Biol, 2017, 40(1 suppl 1):226-237. doi: S1415-47572017000200226 pmid: 28350037
[21]	Alok A, Kaur J, Tiwari S. Functional characterization of wheat myo-inositol oxygenase promoter under different abiotic stress conditions in Arabidopsis thaliana[J]. Biotechnol Lett, 2020, 42(10):2035-2047. doi: 10.1007/s10529-020-02967-1 URL
[22]	Qian WJ, Xiao B, Wang L, et al. CsINV5, a tea vacuolar invertase gene enhances cold tolerance in transgenic Arabidopsis[J]. BMC Plant Biol, 2018, 18(1):228. doi: 10.1186/s12870-018-1456-5 URL

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

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

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

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