利用RNAi技术敲除拟南芥AtCDPK1和AtCDPK1α基因的研究
摘要
钙依赖蛋白激酶(CDPKs)是一个重要的蛋白激酶家族,在植物响应生物和非生物胁迫中起重要作用。拟南芥CDPKs家族中共有34个成员,其中AtCDPK1和AtCDPK1a高度同源。已有的研究结果表明,二者具有相似的功能。基于RNAi技术在基因功能研究中目的性强、周期短的特点,本文利用RNAi技术对AtCDPK1和AtCDPK1a基因进行沉默,并对获得的拟南芥转基因纯合体植株的沉默程度进行了检测,结果如下:
1、针对拟南芥AtCDPK1和AtCDPK1a基因构建了5个RNAi表达载体,其中共沉默RNAi表达载体3个、单一基因沉默的RNAi表达载体各1个。通过蘸花法将其分别转入野生型拟南芥Col-0,获得2个共沉默转基因纯合体和沉默了CDPK1a基因的转基因纯合体;尚有1个共沉默转基因株系和沉默了CDPK1基因的转基因株系已进入T2代筛选。
2、用半定量RT-PCR检测转基因纯合体植株中目的基因沉默程度的结果表明:
(1)CDPK-a的6个株系中两个目的基因的沉默程度都很强。其中,转基因植株10-2的AtCDPK1和AtCDPK1a两个基因的沉默程度最强。
(2)CDPK-c的5个株系中AtCDPK1a的沉默程度比AtCDPK1强。其中,沉默程度最强的是转基因植株2-2。
(3)CDPK1a-2的9个株系中对目的基因AtCDPK1a的沉默程度都较强。其中株系29-4和36-5的沉默程度更强。
3、用半定量RT-PCR检测了转基因纯合体CDPK1a-2的基因沉默特异性,结果表明9个株系都有较高的沉默特异性。
Calcium-dependent protein kinases (CDPKs) belong to an important protein kinase family, and play critical roles in plant responses to biotic and abiotic stress. Among the 34 CDPK members encoded in Arabidopsis genome, AtCDPK1 and AtCDPK1 share high homology. Existing research results showed that these two CDPKs also have similar functions. Take advantages of RNAi technology in specificity and robust on gene family members, it was used in this study to silence AtCDPK1 and AtCDPK1a. The results showed:
1 Totally five RNAi constructs were made. Three constructs were designed to target to different regions of both AtCDPK1 and AtCDPK1a, and two constructs were targeted to AtCDPK1 and AtCDPK1a respectively. The above mentioned constructs were transformed into Arabidopsis Columbia ecotype by flower-dipping method mediated by agrobacteria. Two co-supression constructs and the one targeted to AtCDPK1a had obtained homozygous transgenic lines, the other two constructs are screening the T2 transgenic plants.
2 Using semi-quantitative RT-PCR, the degree of gene silencing in homozygous transgenic plants were checked:
(1) For the co-supression construct CDPK-a, all the six transgenic lines showed high level of silencing on both genes. Among them, line 10-2 showed strongest level of silencing.
(2) For the co-supression construct CDPK-c, silencing level of AtCDPK1a was stronger than AtCDPK1 in all five lines. Among them, line 2-2 has the strongest level silencing.
(3) Nine lines of CDPK1a-2 construct targeting to AtCDPK1a showed specific silecing of the target gene. Lines 29-4 and 36-5 showed much stronger degree of silencing.
3 High specificity of silencing was confirmed by semi-quantitative RT-PCR in transgenic homozygous CDPK1a-2 lines.
1、针对拟南芥AtCDPK1和AtCDPK1a基因构建了5个RNAi表达载体,其中共沉默RNAi表达载体3个、单一基因沉默的RNAi表达载体各1个。通过蘸花法将其分别转入野生型拟南芥Col-0,获得2个共沉默转基因纯合体和沉默了CDPK1a基因的转基因纯合体;尚有1个共沉默转基因株系和沉默了CDPK1基因的转基因株系已进入T2代筛选。
2、用半定量RT-PCR检测转基因纯合体植株中目的基因沉默程度的结果表明:
(1)CDPK-a的6个株系中两个目的基因的沉默程度都很强。其中,转基因植株10-2的AtCDPK1和AtCDPK1a两个基因的沉默程度最强。
(2)CDPK-c的5个株系中AtCDPK1a的沉默程度比AtCDPK1强。其中,沉默程度最强的是转基因植株2-2。
(3)CDPK1a-2的9个株系中对目的基因AtCDPK1a的沉默程度都较强。其中株系29-4和36-5的沉默程度更强。
3、用半定量RT-PCR检测了转基因纯合体CDPK1a-2的基因沉默特异性,结果表明9个株系都有较高的沉默特异性。
Calcium-dependent protein kinases (CDPKs) belong to an important protein kinase family, and play critical roles in plant responses to biotic and abiotic stress. Among the 34 CDPK members encoded in Arabidopsis genome, AtCDPK1 and AtCDPK1 share high homology. Existing research results showed that these two CDPKs also have similar functions. Take advantages of RNAi technology in specificity and robust on gene family members, it was used in this study to silence AtCDPK1 and AtCDPK1a. The results showed:
1 Totally five RNAi constructs were made. Three constructs were designed to target to different regions of both AtCDPK1 and AtCDPK1a, and two constructs were targeted to AtCDPK1 and AtCDPK1a respectively. The above mentioned constructs were transformed into Arabidopsis Columbia ecotype by flower-dipping method mediated by agrobacteria. Two co-supression constructs and the one targeted to AtCDPK1a had obtained homozygous transgenic lines, the other two constructs are screening the T2 transgenic plants.
2 Using semi-quantitative RT-PCR, the degree of gene silencing in homozygous transgenic plants were checked:
(1) For the co-supression construct CDPK-a, all the six transgenic lines showed high level of silencing on both genes. Among them, line 10-2 showed strongest level of silencing.
(2) For the co-supression construct CDPK-c, silencing level of AtCDPK1a was stronger than AtCDPK1 in all five lines. Among them, line 2-2 has the strongest level silencing.
(3) Nine lines of CDPK1a-2 construct targeting to AtCDPK1a showed specific silecing of the target gene. Lines 29-4 and 36-5 showed much stronger degree of silencing.
3 High specificity of silencing was confirmed by semi-quantitative RT-PCR in transgenic homozygous CDPK1a-2 lines.
引文
1 Harper JF, Breton G, et al. Decoding Ca2+ signals through plant protein kinases[J]. Annual Review of Plant Biology, 2004, Vol. 55: 263-288
2 Stone J M. Walker J C. Plant protein kinase families and signal transduction[J] . Plant Physiol . 1995 ,108 :451-457
3 van der Krol AR, Mur LA, et al. Inhibition of flower pigmentation by antisense CHS genes: promoter and minimal sequence requirements for the antisense effect [J]. Plant molecular biology, 1990, 14(4): 457-466
4 Guo S, Kemphues KJ. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed[J]. Cell, 1995, 81(4): 611-620
5 Waterhouse P M, Graham M W, WangM B. Virus resistance and gene silencing in p lants can be induced by simultaneous expression of sense and antisense RNA [J].Proc NatlAcad Sci, 1998, 95: 13959 -13964
6 Fire A, Xu S, Mell o C C, et a1 . Potent and specific genetic interference by double - stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391: 806 - 811
7 Hamilton AJ, Baulcombe DC, A species of small antisense RNA in posttranscriptional gene silencing in plants[J]. Science, 1999, 286(5441): 950-952
8 Sheen J.Ca2+-dependent protein kinases and stress signal transduction in plants[J].Science,1996,274(12):1900-1902
9 Chuang C F,Meyerowitz E M. Specific and heritable genetic interference by double- stranded RNA in Arabidopsis thaliana[J].Proc .Natl.Acad.Sci.2000 ,U.S.A.97:4985-4990
10 Daisuke Miki, Rika It oh, Ko Shi mamoto . RNA silencing of single and multi p le members in a gene family of rice.[J]. Plant Physi ol ogy, 2005, 138: 1903 - 1913
11 Chris Bakal, Rune Linding et al.Phosphorylation Networks Regulating JNK Activity in Diverse Genetic Backgrounds[J]. Science,2008, 322(5900): 453– 456
12 Chioa - Fen Chuang ,Meyerowite EM. Specific and heritable genetic interference by double stranded RNA in Arabidopsis thaliana [J]. Proc Natl Acad Sci VSA,2000, 97: 4985 - 4990 .
13 Meilan R , Branner A M, Skinner J S, et al . Modificati on of flowering intransgenic trees[A]. Morohoahi N ,komamine A. Moleculatr Breeding of woody plant [C].The Netherland: Elsevier Sci Bv . 2001, 41: 247 - 256 .
14 Pinto YM, Kok R A, Bandcombe D C. Resistance to rice yellow mottle virus ( RY MV ) in cultivated African rice varieties containing RY MV transgenes [J]. Nature Biotechnology, 1999, 17: 702-707 .
15 Kusaba M,Miyahara K, Iida S, et al . Low glutelin content1: a dominant mutation that suppresses the glutelin multigene family via RNA silencing in rice [ J ]. Plant Cell, 2003, 15: 1455 - 1467 .
16 Sanders D, Brownlee C, et al. Communicating with calcium [J]. Plant Cell, 1999, Vol. 11: 691-706
17 Rudd J, Franklin-Tong V. Calcium signaling in plants [J]. Cellular and Molecular Life Sciences, 1999, Vol. 55: 214-232
18 Evans NH, McAinsh MR, et al. Calcium oscillations in higher plants [J]. Current Opinion in Plant Biology, 2001, Vol. l4: 415-420
19 Saunders M J,Hepler P K.Calcium ionophore A23187 stimulates cytokinin-like mitosis in Funaria[J].Science,1982,217(9):943-945
20 Hepler P K,Wayne R O.Calcium and Plant Development[J].Annual Review of Plant Physiology,1985,36(6):397-439
21 Ferguson I B,Watkins C B,Jane E.H.Inhibition by Calcium of Senescence of Detached Cucumber Cotyledons:Effect on Ethylene and Hydroperoxide Production[J].Plant Physiol,1993,71(1):182-186
22 Lynch J, Polite VS.lauchli A. Salinity stress increases cytoplasmic Ca activity in maise root protoplasts [J].Plant Physiology. 1989, 90: 1271-1274
23 Haya F,Eliezer E G,Abraham H.Halevy involvement of calcium in the photoperiodic flower induction process of Pharbitis nil[J].Plant Physiol,1989,89(2):530-534
24 Knight MR, Campbell AK, et al.Transgenic Plant aequorun reports the effects of touch and cold-shork and enhance on cytoplasmic calcuim [J]. Nature, 1991, 352: 524-526
25 Liu J,ZuJ K.An Arabidopsis mutant that luires increased calcium for potassium nutrition and salt tolerance[J].Proc Nail Acad Sci U S A .l997.94:l4960-l4964.
26 Lju J , u JK . A Calcium Sensor Homolog Requital for Plant Salt Tolerance[J].Science.1998,280:1943-1945.
27 Hanks S K,Quinn A M.Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members[J].Meth Eezymol,1991,200(7):38-62
28 Walker J C.Structure and function of the receptor-like protein kinases of higher plants[J].Plant Mol Biol,1994,26(5):1599-1609
29 Harmon AC, Putnam-Evans, et al.A calcium-dependent but calmodulin-independent protein kinase from soybean[J]. Plant Physiology 1987, 83: 830-837.
30 Harper J F, Sussman MR et al. A calcium-dependent protein kinase with a regulatorydomain similar to calmodulin[J]. Science, 1991, 252: 951-954
31 Alice C H. Michael G. Jeffrey F H. CDPKs - a kinase for ever Ca2 + signal [J]. Trends in Plant Science , 2000,1:54-159.
32 Cheng SH, Wilmann MR, et al. Calcium signaling through protein kinases.The Arabidopsis calcium-dependent protein kinase gene family [J]. Plant Physiology, 2002, Vol. 129: 469-485
33 Li A L , Zhu Y F , Tan X M , et al . Evolutionary and functional study of the CDPK gene family in wheat ( Triticum aestivum L. ) [J] . Plant Molecular Biology , 2008 , 66 (4) : 429-443.
34 Romeis T , Ludwig A A , Martin R , et al . Calcium-dependent protein kinase play an essential role in a plant defence response[J] .The EMBO Journal , 2001 , 20 : 5556-5567.
35 Yu X C , Zhu S Y, Gao G F , et al . Expression of a grape calcium-dependent protein kinase ACPK1 in A rabi dopsis thal iana promotes plant growth and confers abscisic acid-hypersensitivity in germination , postgermination growth , and stomatal movement [J] . Plant Molecular Biology , 2007 , 64 (5) : 531-538.
36 Kobayashi M , Ohura I , Kawakita K, et al . Calcium dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase[J] . Plant Cell , 2007 , 19 : 1065-1080.
37 Estelle M. Hrabak, Catherine W.M, et al.The Arabidopsis CDPK-SnRK superfamily of protein kinases [J]. Plant Physiology, 2003, Vol. 132: 666-680
38 Klimecka M, Muszyska G. Structure and functions of plant calcium-dependent protein kinases[J]. Acta Biochimica Polonica, 2007, Vol. 54(2): 219-233
39 Harmon AC, Yoo BC, et al. Pseudosubstrate inhibition of CDPK, a protein kinase with a calmodulin-like domain[J]. Bioehemistry 1994, Vol. 33(23): 7278-7287
40 Ulmasov T,Hagen G,Guilfoyle T. The ocs element in the soybean GH2/4 promoter is activated by both active and inactive auxin and salicylic acid analogues[J]. Plant Molecular Biology,1994,26 (4):1055-1064
41 Hong Y,Takano M,Liu C M,Gasch A,Chye M L,Chua N H.Expression of three members of the calcium-dependent protein kinase gene family in Arabidopsis thaliana[J].Plant Mol Biol,1996,30(13):1259-1275
42 Yung X,Deng KQ,et al. A calcium-dependent protein kinase is involved in plant hormone signal transduction in Arabidopsis [J]. Plant Physiology and Biology, 2007, Vol. 33: 227-234
43 Zhang M , Liang S , Lu Y T. Cloning and functional characterization of NtCPK4 , anew tobacco calcium-dependent protein kinase[J] . Biochimica et Biophysica Acta , 2005 , 1729 : 174-185
44 Urao T, Katagiri T, Mizoguchi T. Two gene that encode Ca2+-dependent protein kinase are induced by drought and high salt stress in Arabidopsis thaliana[J]. Mol Gen Genet 1994,244 :331-340
45 Anil VS,Harmon AC,et al. Temporal association of Ca2+-dependent protein kinase with oi1 bodies during seed development in Santalum album L. its biochemical characterization and significance [J]. Plant Cell Physiology, 2003, Vol. 44(4): 367-376
46 Davletova S,Meszaros T,et al. Auxin and heat shock activation of a novel member of the calmodulin-like domain protein kinase gene family in cultured alfalfa cells [J]. Journal of Experimental Botany. 2001, 52, 215-221
47 Lee J , Rudd J J . Calcium - dependent protein kinases : versatile plant signalling components necessary for pathogen defence[J]. Trends in Plant Science. 2002 ,7 :97 - 98.
48 Chung E, Park JM, et al. Molecular and biochemical characterization of the Capsicum annuum calcium-dependent protein kinase 3 (CaCDPK3) gene induced by abiotic and biotic stresses [J]. Planta, 2004, Vol. 220:286-295
49 Chico JM, Raíces M,et al. A calcium-dependent protein kinase is systemically induced upon wounding in tomato plants [J]. Plant Physiology, 2002, Vol. 128: 256-270
50 Xing T,Higgins VJ,et al. Regulation of plant defense response to fungal pathogens: Two types of protein kinases in the reversible phosphorylation of the host plasma membrane H+-ATPase [J]. Plant Cell, 1996, Vol. 8: 555-564
51 Murillo I, Jaeck E, et al. Transcriptional activation of a maize calcium-dependent protein kinase gene in response to fungal elicitor and infection [J]. Plant Molecular Biology, 2001, Vol. 45: 145-158
52 Zhu SY, Yu XC, et al. Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidopsis [J]. Plant Cell, 2007, Vol. 19: 3019-3036
53 Gyeong Mee Yoon, Peter E Dowd, Simon Gilroy, et al. Calcium-dependent protein kinase isoforms in petunia have distinct functions in pollen tube growth, Including regulating polarity [J]. Plant Cell, 2006, 18: 867-878
54卢彗星.拟南芥AtCDPK1参与非生物胁迫响应和ABA信号途径的证据.内蒙古农业大学硕士学位论文
55潘瑞炽.植物组织培养[M],广东高等教育出版社, 2000
56 Murray MG, Thompson WF. Rapid isolation of high molecular weight plant DNA [J].Nucleic Acids Research. 1980, 8(19): 4321-4325
57 Clough SJ, Bent A F. Floral dip. a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana [J]. The Plant Journal, 1998, 16(6): 735-743
58 S. Varsha Wesley, Christopher A. Helliwell, et al.Construct design for effcient, effective and highthroughput gene silencing in plants[J]. The Plant Journal 2001, 27(6):581-590
2 Stone J M. Walker J C. Plant protein kinase families and signal transduction[J] . Plant Physiol . 1995 ,108 :451-457
3 van der Krol AR, Mur LA, et al. Inhibition of flower pigmentation by antisense CHS genes: promoter and minimal sequence requirements for the antisense effect [J]. Plant molecular biology, 1990, 14(4): 457-466
4 Guo S, Kemphues KJ. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed[J]. Cell, 1995, 81(4): 611-620
5 Waterhouse P M, Graham M W, WangM B. Virus resistance and gene silencing in p lants can be induced by simultaneous expression of sense and antisense RNA [J].Proc NatlAcad Sci, 1998, 95: 13959 -13964
6 Fire A, Xu S, Mell o C C, et a1 . Potent and specific genetic interference by double - stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391: 806 - 811
7 Hamilton AJ, Baulcombe DC, A species of small antisense RNA in posttranscriptional gene silencing in plants[J]. Science, 1999, 286(5441): 950-952
8 Sheen J.Ca2+-dependent protein kinases and stress signal transduction in plants[J].Science,1996,274(12):1900-1902
9 Chuang C F,Meyerowitz E M. Specific and heritable genetic interference by double- stranded RNA in Arabidopsis thaliana[J].Proc .Natl.Acad.Sci.2000 ,U.S.A.97:4985-4990
10 Daisuke Miki, Rika It oh, Ko Shi mamoto . RNA silencing of single and multi p le members in a gene family of rice.[J]. Plant Physi ol ogy, 2005, 138: 1903 - 1913
11 Chris Bakal, Rune Linding et al.Phosphorylation Networks Regulating JNK Activity in Diverse Genetic Backgrounds[J]. Science,2008, 322(5900): 453– 456
12 Chioa - Fen Chuang ,Meyerowite EM. Specific and heritable genetic interference by double stranded RNA in Arabidopsis thaliana [J]. Proc Natl Acad Sci VSA,2000, 97: 4985 - 4990 .
13 Meilan R , Branner A M, Skinner J S, et al . Modificati on of flowering intransgenic trees[A]. Morohoahi N ,komamine A. Moleculatr Breeding of woody plant [C].The Netherland: Elsevier Sci Bv . 2001, 41: 247 - 256 .
14 Pinto YM, Kok R A, Bandcombe D C. Resistance to rice yellow mottle virus ( RY MV ) in cultivated African rice varieties containing RY MV transgenes [J]. Nature Biotechnology, 1999, 17: 702-707 .
15 Kusaba M,Miyahara K, Iida S, et al . Low glutelin content1: a dominant mutation that suppresses the glutelin multigene family via RNA silencing in rice [ J ]. Plant Cell, 2003, 15: 1455 - 1467 .
16 Sanders D, Brownlee C, et al. Communicating with calcium [J]. Plant Cell, 1999, Vol. 11: 691-706
17 Rudd J, Franklin-Tong V. Calcium signaling in plants [J]. Cellular and Molecular Life Sciences, 1999, Vol. 55: 214-232
18 Evans NH, McAinsh MR, et al. Calcium oscillations in higher plants [J]. Current Opinion in Plant Biology, 2001, Vol. l4: 415-420
19 Saunders M J,Hepler P K.Calcium ionophore A23187 stimulates cytokinin-like mitosis in Funaria[J].Science,1982,217(9):943-945
20 Hepler P K,Wayne R O.Calcium and Plant Development[J].Annual Review of Plant Physiology,1985,36(6):397-439
21 Ferguson I B,Watkins C B,Jane E.H.Inhibition by Calcium of Senescence of Detached Cucumber Cotyledons:Effect on Ethylene and Hydroperoxide Production[J].Plant Physiol,1993,71(1):182-186
22 Lynch J, Polite VS.lauchli A. Salinity stress increases cytoplasmic Ca activity in maise root protoplasts [J].Plant Physiology. 1989, 90: 1271-1274
23 Haya F,Eliezer E G,Abraham H.Halevy involvement of calcium in the photoperiodic flower induction process of Pharbitis nil[J].Plant Physiol,1989,89(2):530-534
24 Knight MR, Campbell AK, et al.Transgenic Plant aequorun reports the effects of touch and cold-shork and enhance on cytoplasmic calcuim [J]. Nature, 1991, 352: 524-526
25 Liu J,ZuJ K.An Arabidopsis mutant that luires increased calcium for potassium nutrition and salt tolerance[J].Proc Nail Acad Sci U S A .l997.94:l4960-l4964.
26 Lju J , u JK . A Calcium Sensor Homolog Requital for Plant Salt Tolerance[J].Science.1998,280:1943-1945.
27 Hanks S K,Quinn A M.Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members[J].Meth Eezymol,1991,200(7):38-62
28 Walker J C.Structure and function of the receptor-like protein kinases of higher plants[J].Plant Mol Biol,1994,26(5):1599-1609
29 Harmon AC, Putnam-Evans, et al.A calcium-dependent but calmodulin-independent protein kinase from soybean[J]. Plant Physiology 1987, 83: 830-837.
30 Harper J F, Sussman MR et al. A calcium-dependent protein kinase with a regulatorydomain similar to calmodulin[J]. Science, 1991, 252: 951-954
31 Alice C H. Michael G. Jeffrey F H. CDPKs - a kinase for ever Ca2 + signal [J]. Trends in Plant Science , 2000,1:54-159.
32 Cheng SH, Wilmann MR, et al. Calcium signaling through protein kinases.The Arabidopsis calcium-dependent protein kinase gene family [J]. Plant Physiology, 2002, Vol. 129: 469-485
33 Li A L , Zhu Y F , Tan X M , et al . Evolutionary and functional study of the CDPK gene family in wheat ( Triticum aestivum L. ) [J] . Plant Molecular Biology , 2008 , 66 (4) : 429-443.
34 Romeis T , Ludwig A A , Martin R , et al . Calcium-dependent protein kinase play an essential role in a plant defence response[J] .The EMBO Journal , 2001 , 20 : 5556-5567.
35 Yu X C , Zhu S Y, Gao G F , et al . Expression of a grape calcium-dependent protein kinase ACPK1 in A rabi dopsis thal iana promotes plant growth and confers abscisic acid-hypersensitivity in germination , postgermination growth , and stomatal movement [J] . Plant Molecular Biology , 2007 , 64 (5) : 531-538.
36 Kobayashi M , Ohura I , Kawakita K, et al . Calcium dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase[J] . Plant Cell , 2007 , 19 : 1065-1080.
37 Estelle M. Hrabak, Catherine W.M, et al.The Arabidopsis CDPK-SnRK superfamily of protein kinases [J]. Plant Physiology, 2003, Vol. 132: 666-680
38 Klimecka M, Muszyska G. Structure and functions of plant calcium-dependent protein kinases[J]. Acta Biochimica Polonica, 2007, Vol. 54(2): 219-233
39 Harmon AC, Yoo BC, et al. Pseudosubstrate inhibition of CDPK, a protein kinase with a calmodulin-like domain[J]. Bioehemistry 1994, Vol. 33(23): 7278-7287
40 Ulmasov T,Hagen G,Guilfoyle T. The ocs element in the soybean GH2/4 promoter is activated by both active and inactive auxin and salicylic acid analogues[J]. Plant Molecular Biology,1994,26 (4):1055-1064
41 Hong Y,Takano M,Liu C M,Gasch A,Chye M L,Chua N H.Expression of three members of the calcium-dependent protein kinase gene family in Arabidopsis thaliana[J].Plant Mol Biol,1996,30(13):1259-1275
42 Yung X,Deng KQ,et al. A calcium-dependent protein kinase is involved in plant hormone signal transduction in Arabidopsis [J]. Plant Physiology and Biology, 2007, Vol. 33: 227-234
43 Zhang M , Liang S , Lu Y T. Cloning and functional characterization of NtCPK4 , anew tobacco calcium-dependent protein kinase[J] . Biochimica et Biophysica Acta , 2005 , 1729 : 174-185
44 Urao T, Katagiri T, Mizoguchi T. Two gene that encode Ca2+-dependent protein kinase are induced by drought and high salt stress in Arabidopsis thaliana[J]. Mol Gen Genet 1994,244 :331-340
45 Anil VS,Harmon AC,et al. Temporal association of Ca2+-dependent protein kinase with oi1 bodies during seed development in Santalum album L. its biochemical characterization and significance [J]. Plant Cell Physiology, 2003, Vol. 44(4): 367-376
46 Davletova S,Meszaros T,et al. Auxin and heat shock activation of a novel member of the calmodulin-like domain protein kinase gene family in cultured alfalfa cells [J]. Journal of Experimental Botany. 2001, 52, 215-221
47 Lee J , Rudd J J . Calcium - dependent protein kinases : versatile plant signalling components necessary for pathogen defence[J]. Trends in Plant Science. 2002 ,7 :97 - 98.
48 Chung E, Park JM, et al. Molecular and biochemical characterization of the Capsicum annuum calcium-dependent protein kinase 3 (CaCDPK3) gene induced by abiotic and biotic stresses [J]. Planta, 2004, Vol. 220:286-295
49 Chico JM, Raíces M,et al. A calcium-dependent protein kinase is systemically induced upon wounding in tomato plants [J]. Plant Physiology, 2002, Vol. 128: 256-270
50 Xing T,Higgins VJ,et al. Regulation of plant defense response to fungal pathogens: Two types of protein kinases in the reversible phosphorylation of the host plasma membrane H+-ATPase [J]. Plant Cell, 1996, Vol. 8: 555-564
51 Murillo I, Jaeck E, et al. Transcriptional activation of a maize calcium-dependent protein kinase gene in response to fungal elicitor and infection [J]. Plant Molecular Biology, 2001, Vol. 45: 145-158
52 Zhu SY, Yu XC, et al. Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidopsis [J]. Plant Cell, 2007, Vol. 19: 3019-3036
53 Gyeong Mee Yoon, Peter E Dowd, Simon Gilroy, et al. Calcium-dependent protein kinase isoforms in petunia have distinct functions in pollen tube growth, Including regulating polarity [J]. Plant Cell, 2006, 18: 867-878
54卢彗星.拟南芥AtCDPK1参与非生物胁迫响应和ABA信号途径的证据.内蒙古农业大学硕士学位论文
55潘瑞炽.植物组织培养[M],广东高等教育出版社, 2000
56 Murray MG, Thompson WF. Rapid isolation of high molecular weight plant DNA [J].Nucleic Acids Research. 1980, 8(19): 4321-4325
57 Clough SJ, Bent A F. Floral dip. a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana [J]. The Plant Journal, 1998, 16(6): 735-743
58 S. Varsha Wesley, Christopher A. Helliwell, et al.Construct design for effcient, effective and highthroughput gene silencing in plants[J]. The Plant Journal 2001, 27(6):581-590