巴西橡胶树硫氧还蛋白基因HbTRX的克隆与表达分析
摘要
巴西橡胶树(Hevea brasiliensis)是天然橡胶的主要来源。巴西橡胶树的幼态无性系具有生长速度快、产胶量高、抗逆性强的特点,是一种具有很大应用潜力的新型种植材料。对橡胶树幼态无性系高产的机制还不清楚,目前仅限于对幼态无性系和老态无性系之间差异的形态学观察、排胶生理参数等的研究,而缺乏直接的分子生物学、遗传学等方面的证据,在一定程度上限制了橡胶树幼态无性系在生产中的应用和大面积推广。因此开展橡胶树幼态无性系高产的分子基础研究将对我们进一步了解橡胶树幼态无性系增产的原因具有重要的理论意义；同时对推动橡胶树幼态无性系种植材料的推广和应用,提高天然橡胶的产量也将具有重要的实践意义。
本课题组通过抑制缩减杂交(SSH)获得一个在幼态无性系和老态无性系之间差异表达的片段(HbSSH),经过BlastX分析,发现HbSSH所编码的氨基酸序列与蓖麻(Ricinus communis)硫氧还蛋白氨基酸序列的同源性很高。本研究根据HbSSH的序列信息设计引物,通过RT-PCR和RACE技术从巴西橡胶的胶乳中获得了目的基因(命名为HbTRX),该基因总长546bp,其中包括30bp的5'-UTR和144bp的3'-UTR,编码123个氨基酸,该氨基酸序列与蓖麻、毛果杨、葡萄、轮叶党参、拟南芥、大球果黄杉的TRX氨基酸序列同源性达到了79%、78%、77%、77%、72%、69%。生物信息学表明HbTRX具有TRX典型的WCXPC结构域。
半定量RT-PCR结果显示,HbTRX基因的表达具有组织差异性,在愈伤中大量表达,胶乳中次之,在花、叶、芽、树皮中均微量表达；它在橡胶树733、海2、海1三种品系的老态无性系和幼态无性系胶乳中均有差异表达,均为老态无性系胶乳中较幼态胶乳中高表达；乙烯和茉莉酸对胶乳HbTRX基因的表达几乎没有影响。构建HbTRX的原核表达载体pET-30a-HbTRX,并在大肠杆菌BL21(DE3)中进行了表达,经过IPTG诱导4个小时之后,在SDS-PAGE中显示出特异性条带,产生包括His-tag(约5.4 kD)在内的约21.3 kD的重组HbTRX蛋白,和理论上蛋白的大小基本一致。超声波破碎细胞后,经SDS-PAGE检测发现重组蛋白在大肠杆菌BL21中是以可溶蛋白的形式存在的。
为了进一步探究HbTRX的特性和功能,本研究采用Ni2+螯合亲和层析法分离出重组HbTRX蛋白,并构建了两组反应体系,第一组为25μg／ml重组HbTRX蛋白与牛胰岛素构成的反应体系,第二组为50μg／ml重组HbTRX蛋白与牛胰岛素构成的反应体系,这两组体系均有DTT的参与。结果显示,这两组反应均在650nm的光波下显示出逐渐上升的光吸收值,而50μg／ml重组HbTRX蛋白与牛胰岛素构成的反应体系的光吸收值更高,90min之后这两组反应均进入平台期。这个结果表明,重组HbTRX能降低牛胰岛素二硫化物的结合,具有氧化还原活性。
Hevea brasiliensis is the main source of natural rubber. The juvenile clone of Hevea brasiliensis has great potential a pplication as a new type of planting material, with fast growth speed, high volume production of rubber, and the advantages of strong resistance. Now the research on morphology, physiology and cultivation of Hevea brasiliensis juvenile clone had made considerable progress, and the volume production of rubber reached a certain level. But there is no sufficient evidence of molecular biology to directly prove its high-yielding mechanism. The molecular biology research of rubber is still backward, thus limited the production applications and large-scale promotion of Hevea brasiliensis juvenile clone.
So it has important theoretical and practical significance to do some work on the biological function of differentially expressed genes in rubber latex of old and juvenile clone of Hevea brasiliensis. A differentially expressed fragment of latex in old and juvenile clone of Hevea brasiliensis was obtained through the suppression subtractive hybridization by our reseach group.It encodes a thioredoxin-like amino acid analyzed by BlastX. Because thioredoxin is a class of multi-functional acidic protein existed in vivo and can regulate the activity of some key enzymes during the photosynthesis process in plant. The study of thioredoxin in rubber tree is shortage, and its function is not clear, therefore, by using the information about the acquired sequence (HbSSH), the thioredoxin was cloned by the technology of RT-PCR and RACE, named HbTRX. Sequence analysis revealed that the ORF of HbTRX encodes 123 amino acid. The deduced amino acid sequences of HbTRX showed high identities (79%,78%,77%,77%,72%,69%) to those of thioredoxin from Ricinus communis, Populus trichocarpa, Vitis vinifera, Codonopsis lanceolata, Arabidopsis thaliana and Pseudotsuga macrocarpa. Bioinformatics shows that HbTRX was a member of TRX-family, with a similar CXXC domain.
Semi-quantitative RT-PCR analysis indicates that the HbTRX is most highly expressed in callus, and more highly expressed in latex than in flower, in enbryos, in leaf and bud. And it is highly expressed in old clone of every type of rubber tree. The transcription of HbTRX was not induced in the latex by jasmonic acid and ethylene. In order to further study the function of HbTRX, construct HbTRX prokaryotic expression vector pET-30a-HbTRX, specific protein band was detected by SDS-PAGE in the cell homogenate by induction with IPTG in the E.coli cells harboring pET-30a-HbTRX produced recombinant proteins with a total predicted molecular mass of 21.296kD. The recombinant proteins is a form of soluble protein. Ultrasonic broked cells with recombinant proteins. The fact was that recombinant protein was a soluble protein in E. coli (BL21) by SDS-PAGE.
In order to understanding the features and functionality of HbTRX protein, the study separated recombinant proteins by Ni2+ chelate affinity chromatography, and constructed two reaction system with the presence of DTT. The first reaction system is composed of 25μg/ml recombinant protein and bovine insulin, the second is composed of 50μg/ml recombinant protein and bovine insulin. The results showed that, the two groups displayed that the light absorption value was gradually increased under in the 650nm light waves. The light absorption value of the second reaction was higher than the first reaction, and both of reaction groups went into plateau 90min later. This shows that, the recombinant protein in E. coli, can reduce bovine insulin disulfides bridges, with weak redox activity.
本课题组通过抑制缩减杂交(SSH)获得一个在幼态无性系和老态无性系之间差异表达的片段(HbSSH),经过BlastX分析,发现HbSSH所编码的氨基酸序列与蓖麻(Ricinus communis)硫氧还蛋白氨基酸序列的同源性很高。本研究根据HbSSH的序列信息设计引物,通过RT-PCR和RACE技术从巴西橡胶的胶乳中获得了目的基因(命名为HbTRX),该基因总长546bp,其中包括30bp的5'-UTR和144bp的3'-UTR,编码123个氨基酸,该氨基酸序列与蓖麻、毛果杨、葡萄、轮叶党参、拟南芥、大球果黄杉的TRX氨基酸序列同源性达到了79%、78%、77%、77%、72%、69%。生物信息学表明HbTRX具有TRX典型的WCXPC结构域。
半定量RT-PCR结果显示,HbTRX基因的表达具有组织差异性,在愈伤中大量表达,胶乳中次之,在花、叶、芽、树皮中均微量表达；它在橡胶树733、海2、海1三种品系的老态无性系和幼态无性系胶乳中均有差异表达,均为老态无性系胶乳中较幼态胶乳中高表达；乙烯和茉莉酸对胶乳HbTRX基因的表达几乎没有影响。构建HbTRX的原核表达载体pET-30a-HbTRX,并在大肠杆菌BL21(DE3)中进行了表达,经过IPTG诱导4个小时之后,在SDS-PAGE中显示出特异性条带,产生包括His-tag(约5.4 kD)在内的约21.3 kD的重组HbTRX蛋白,和理论上蛋白的大小基本一致。超声波破碎细胞后,经SDS-PAGE检测发现重组蛋白在大肠杆菌BL21中是以可溶蛋白的形式存在的。
为了进一步探究HbTRX的特性和功能,本研究采用Ni2+螯合亲和层析法分离出重组HbTRX蛋白,并构建了两组反应体系,第一组为25μg／ml重组HbTRX蛋白与牛胰岛素构成的反应体系,第二组为50μg／ml重组HbTRX蛋白与牛胰岛素构成的反应体系,这两组体系均有DTT的参与。结果显示,这两组反应均在650nm的光波下显示出逐渐上升的光吸收值,而50μg／ml重组HbTRX蛋白与牛胰岛素构成的反应体系的光吸收值更高,90min之后这两组反应均进入平台期。这个结果表明,重组HbTRX能降低牛胰岛素二硫化物的结合,具有氧化还原活性。
Hevea brasiliensis is the main source of natural rubber. The juvenile clone of Hevea brasiliensis has great potential a pplication as a new type of planting material, with fast growth speed, high volume production of rubber, and the advantages of strong resistance. Now the research on morphology, physiology and cultivation of Hevea brasiliensis juvenile clone had made considerable progress, and the volume production of rubber reached a certain level. But there is no sufficient evidence of molecular biology to directly prove its high-yielding mechanism. The molecular biology research of rubber is still backward, thus limited the production applications and large-scale promotion of Hevea brasiliensis juvenile clone.
So it has important theoretical and practical significance to do some work on the biological function of differentially expressed genes in rubber latex of old and juvenile clone of Hevea brasiliensis. A differentially expressed fragment of latex in old and juvenile clone of Hevea brasiliensis was obtained through the suppression subtractive hybridization by our reseach group.It encodes a thioredoxin-like amino acid analyzed by BlastX. Because thioredoxin is a class of multi-functional acidic protein existed in vivo and can regulate the activity of some key enzymes during the photosynthesis process in plant. The study of thioredoxin in rubber tree is shortage, and its function is not clear, therefore, by using the information about the acquired sequence (HbSSH), the thioredoxin was cloned by the technology of RT-PCR and RACE, named HbTRX. Sequence analysis revealed that the ORF of HbTRX encodes 123 amino acid. The deduced amino acid sequences of HbTRX showed high identities (79%,78%,77%,77%,72%,69%) to those of thioredoxin from Ricinus communis, Populus trichocarpa, Vitis vinifera, Codonopsis lanceolata, Arabidopsis thaliana and Pseudotsuga macrocarpa. Bioinformatics shows that HbTRX was a member of TRX-family, with a similar CXXC domain.
Semi-quantitative RT-PCR analysis indicates that the HbTRX is most highly expressed in callus, and more highly expressed in latex than in flower, in enbryos, in leaf and bud. And it is highly expressed in old clone of every type of rubber tree. The transcription of HbTRX was not induced in the latex by jasmonic acid and ethylene. In order to further study the function of HbTRX, construct HbTRX prokaryotic expression vector pET-30a-HbTRX, specific protein band was detected by SDS-PAGE in the cell homogenate by induction with IPTG in the E.coli cells harboring pET-30a-HbTRX produced recombinant proteins with a total predicted molecular mass of 21.296kD. The recombinant proteins is a form of soluble protein. Ultrasonic broked cells with recombinant proteins. The fact was that recombinant protein was a soluble protein in E. coli (BL21) by SDS-PAGE.
In order to understanding the features and functionality of HbTRX protein, the study separated recombinant proteins by Ni2+ chelate affinity chromatography, and constructed two reaction system with the presence of DTT. The first reaction system is composed of 25μg/ml recombinant protein and bovine insulin, the second is composed of 50μg/ml recombinant protein and bovine insulin. The results showed that, the two groups displayed that the light absorption value was gradually increased under in the 650nm light waves. The light absorption value of the second reaction was higher than the first reaction, and both of reaction groups went into plateau 90min later. This shows that, the recombinant protein in E. coli, can reduce bovine insulin disulfides bridges, with weak redox activity.
引文
1.陈三凤.几丁质酶研究历史和发展前景[J].微生物通报,1993,20(6)：156-160.
2.陈守才,邵寒霜,胡东琼,等.用RAPD技术鉴定橡胶树抗白粉病基因连锁标记[J].热带作物学报,1994,15(2)：21-26.
3.陈雄庭,王泽云,吴蝴蝶,张秀娟.橡胶树新种植材料-幼态自根无性系[J],热带作物学报,2002,23(1)：19-23.
4.陈雄庭.橡胶速生高产新型种植材料的培育与试种.2005,中国热带作物学会2005年学术(青年学术)研讨会
5.郝秉忠,吴继林.巴西橡胶树幼态无性系的高产特性[J].热带农业科学,1996,2：1-8.
6. 何波.橡胶树的幼态老态与全息胚理论[J].热带农业科学,1991,04：002.
7.何康,黄宗道.热带北缘橡胶栽培.[M].广州：广东科学技术出版社.1987
8.黄炎,郭庆水,徐立新,陈守才等.巴西橡胶树的分子生物学研究进展[J].安徽农业科学,2008,36(2)：442-445-
9.李守岭,庄南生.植物花药培养及其影响因素研究进展[J].亚热带植物科学,2006,35(3)：76-80
10.梁小莲,李辉亮,彭世清.巴西橡胶树HbTCTP基因的克隆及表达[J].分子植物育种,2009,7(1)：194-198.
11.刘松泉.巴西橡胶树幼态无性系的性质和利用[J].热带作物学术讨论会科技资料汇编,1979,13-20.
12.刘雷,尹钧,硫氧还蛋白的研究[J].东北农业大学学报,2003,34(2)：219-225.
13.鲁惠中,李辉亮,郭冬,彭世清.巴西橡胶树HbHDTl基因的克隆及表达分析[J].热带作物学报,2010,31(1)：59～64.
14.罗安定,陈守才,吴坤鑫等.AFLP在橡胶树优异种质研究中的应用[J].植物学报,2001,43(9)：941-947.
15.彭世清,吴坤金,陈守才.橡胶延长因子的cDNA的克隆和序列分析[J].华南热带农业大学学报,2000,6(2)：2-6
16.彭世清,傅湘辉,吴坤鑫,等.巴西橡胶树死皮病相关基因HbMyb1的结构分析及表达[J].植物生理与分子生物学学报,2003,29(2)：147-152.
17.潘衍庆主编.中国热带作物栽培学[M].北京：中国农业出版社,1998,3-5.
18.宿迁林业园林网.林木无性繁殖研究进展,http://lyj.suqian.gov.cn.
19.王颖,陈雄庭,张秀娟等.基因枪法将GAI基因导入巴西橡胶的研究[J].热带亚热带植物学报,2006,14(3)：179-182.
20.卫志明.花药和花粉培养.植物生理与分子生物学(第二版)[M].北京：科学出版社,1998,16-29.
21.许闻献.中国橡胶树割制改革30年[J].热带农业科学,2000,6：57-71.
22.杨立青-橡胶幼态的获得与利用[J].热带农业科技,2003,26(3)：16-18.
23.幼态课题组.幼态无性系在生产上的应用[J].热带农业科学,1994,2,1-3.
24.于俊红,黄绵佳,田维敏.巴西橡胶树橡胶生物合成调控的研究进展[J].安徽农学通报,2007,13(12)：38-40.
25.张治礼,郑学勤.树木发育阶段理论及其在橡胶树育种中的应用[J].热带亚热带植物学报,2001,9(3)：262-268.
26.中国橡胶网.中国成为越南天然橡胶主要出口国.http://www.cria.org.cn
27.中国橡胶市场网.中国橡胶工业协会会长范仁德讲话.2010,http://www.ex-rubber.com.
28.郑琼,马旭俊,杨传平.硫氧还蛋白(TRX)的研究进展[J].分子植物育种,2006,6(S)：78-82
29.曾日中,段翠芳,黎瑜,郝秉中.茉莉酸刺激的橡胶树胶乳cDNA消减文库的构建及其序列分析[J].热带作物学报,2003,(3)：34-37.
30. Arne Holmgren. Thioredoxin Catalyzes the Reduction of Insulin Disulfides by Dithiothreitol and Dihydrolipoamide[J].The journal of biological chemistry,1979, 254:9627-9632.
31. Arner E S J, Holmgren A. The thioredoxin system in cancer [J]. Seminars in Cancer Biology,2006,16:202-226.
32. Arokiaraj P, Jones H, Cheong K F, et al. Gene insertion into Hevea brasiliensis[J]. Plant Cell Reports,1994,13:425-426
33. Baker A, Payne C M, Briehl M M, et al. Thioredoxin,a gene found overexpressed in human cancer, inhibits apoptosis invitro and in vivo[J]. Cancer Res,1997,57:5162-5167.
34. Besse P, Lebrun P, Seguin M, et al. DNA fingerprints in Hevea brasiliensis using human minisatellite probes [J]. Heredity,1993,70:237-244.
35. Blomback M, Bjarke B,Herin P, et al. Neonatal aortic thrombosis.A possible clinical manifestation of congenital antithrombin III deficiency [J]. Acta Paediatr Scand,1977,63:297-301,1974
36. Broin M and Rey P. Potato plants lacking the CDSP32 plastidic thioredoxin exhibit overoxidation of the BAS12-cysteine peroxiredoxin and increased lipid Peroxidation in thylakoids under photooxidative stress [J]. Plant Physiol,2003,
132:1335-1343
37. Carvalho A P, Fernandes P A, Ramos M J. Similarities and differences in the thioredoxin superfamily[J].Progress in Biophysics and Molecular Biology,2006, 91:229-248.
38. Cornish K, Backhaus R A. Rubber transferase activity in rubber particles of guayule[J].Phytochemistry,1990,29:3809-3813
39. d'Auzac J, Jacob JL, Prevot JC, et al. The regulati on of cis-polyisoprene producti on (natural rubber) from Hevea brasiliensis [J]. Recent Res Plant Physiol, 1997,1:273-331
40. Deng X D, Fei X W, Huang J S. Isolation and analysis of rubber hevein gene and its promoter sequence [J]. Acta Botanica Sinica,2002,44:936-940.
41. Gelhaye E, Rouhier N, Navrot N, and Jacquot J P. The plant thioredoxin system [J]. Life Sci,2005,62:24-35
42. Goyvaerts E, Dennis M, Light D, et al. Cloning and sequencing of the cDNA encoding the rubber elongation factor of Hevea brasiliensis [J]. Plant Physiology, 1991,97:317-321
43. Hao Bingzhong, Wu Jilin. Laticifer differentiation in Hevea brasiliensis: Induced by exogenous jas monic acid and linolenic acid [J]. Annals of Botany, 2000,85:37-43
44. Holmgren A. Hydrogen donor system for Escherichia coli ribonucleoside-diphosphate reductase dependent upon glutathione [J]. Acad. Sci.,1976,73: 2275-2279.
45. Holmgren A. Thioredoxin:The amino acid sequence of the protein from Escherichia coli [J]. Biochem,1968,6:475-484.
46. Holmgren A. Thioredoxin and glutaredoxin systems [J]. Biol Chem,1989,264: 13963-13966.
47. Holmgren, A. Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide [J]. Biol. Chem,1979,254:9627-9632
48. Holmgren A, Bjornstedt M. Enzymatic reduction-oxidation of protein disulfides by thioredoxin [J]. Methods Enzymol,1984,107:295-300.
49. Holmgren J, Lindblad H, Fredman P, et al. Comparison of receptors for cholera and Escherichia coli enterotoxins in human intestine [J]. Gastroenterology,1985, 89:27-35.
50. Holmgren C, Johansson C, Berndt M E, et al. Thioredox control via thioredoxin
and glutaredoxin systems [J]. Cellular Information Processing,2005,1375-1377.
51. Jaglo K R, Gilmours J, Zarka D G, et al. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing to lerance [J]. Science,1998,280: 104-106.
52. Javier Andres Juarez-Dlaz, Bruce McClure, Sonia Vazquez-Santana. A Novel Thioredoxin h Is Secreted in Nicotiana alata and Reduces S-RNase in Vitro [J]. The journal of biological chemistry,2006,281:3418-3424.
53. Juttner J, Olde D, Langridge P, et al. Cloning and expression of a distinct subclass of plant thioredoxins [J]. European Journal of Biochemistry,2000,267: 7109-7117.
54. Kim Y B, Garbisu C, Pickering L J, et al. Thioredoxinh overexpressed in barley seeds enhances selenite resistance and uptake during germination and early seedling development [J]. Planta,2003,218:186-191.
55. Laurent T C, Moore E C, Reichard P, et al. Enzymatic synthesis of deoxyribonucleotides.Ⅳ.Isolation and characterization of thioredoxin, the hydrogen donor from Escherichia coli [J]. Biol.Chem,1964,239:3436-3444.
56. Lee K K, Murakawa M, Takahashi S et al. Purification, molecule cloning, and characterization of TRX32, a novel thioredoxin-related mammalian protein of 32 kD [J]. Biol Chem,1998,273:19160-19166.
57. Light D R, Lazarus R A, Dennis M S et al. Rubber elongation by farnesyl pyrophosphate synthases involves a novel switch in enzymes tereospecificity [J]. The Journal of Biological Chemistry,1989,264:18598-18607.
58. Low F C, Safiah A, Hafsah J, et al. Recent advance in the development of molecular markers for Hevea brasiliensis [J].Journal of Natural Rubber Research, 1996,11:32-44.
59. Mannervik B, Guthenberg C, Jensson H, et al. Isozymes of glutathione S-transferases in rat and human tissues, in Functions of Glutathione:Biochemical, Physiological, Toxicological, and Clinical Aspects [J]. Larsson A, Orrenius S, Holmgren A, and Mannervik B eds,1983,1:75-88.
60. Martin J L. Thioredoxin2a fold for all reasons [J].Structure,1995,3:2452250.
61. Miao Z H, John J G Molecularcloning, characterization and expression of Mn superoxide dismutase from the rubber tree(Hevea brasiliensis) [J]. Plant Molecular Biology,1993,23:267-277.
62. Okada K, Sato T, Nakagawa M, et al. Five geranylgeranyldiphosphate synthases expressed in different organs are localized into three subcellular compartments in Arabidopsis [J]. Plant Physiology,2000,122:1045-1056.
63. Roland Cazalis, Pablo Pulido, Thierry Aussenac, et al. Cloning and characterization of three thioredoxin h isoforms from wheat showing differential expression in seeds [J]. Journal of Experimental Botany,2006,57:2165-2172.
64. Sano H, Sata T, Nanri H, et al. Thioredoxin is associ2 ated with endotoxin tolerance in mice [J]. Crit Care Med,2002,30:192-194.
65. Sederberg A, Sahaf B, Rosn A et al. Thioredoxin reductase, a redox active selenoprotein, is secreted by normal and neoplastic cells:presence in human plasma [J]. Cancer Res,2000,60:2281-2289.
66. Southorn W A. Physiology of Hevea latex flow [J]. Rubber Res,1969,21: 494-512.
67. Southorn W A, Yip E. Latex flow studies III Electrostatic considerations in colloidal stability of fresh Hevea latex [J]. Rubber Res,1968,20:210-215.
68. Tang C, Qi J, Li H, et al. A convenient and efficient protocol for isolating high-quality RNA from latex of Hevea brasiliensis (para rubber tree) [J]. Biochem Biophys Methods,2007,70:749～754.
69. Tian WeiMin, Shi MinJing, Wu JiLin, et al. Localized Effects of mechanical wounding an exogenous jasmonic acid on the induction of secondary laticifer differentiati on in relation to the distribution of jasmonic acid in Hevea brasiliensis[J].Acta Botanica Sinica,2003,45:1366-1372.
70. Ueno M, Masutani H, Arai R J, et al. Thioredoxin dependent redox regulation of p532mediated p21 activation [J]. Biol Chem,2000,274:35809-35815.
71. Venkata C P, Priya P, Sarawathy A C, et al. Identification, cloning and sequence analysis of a dwarf genome specific RAPD marker in rubber tree [J]. Plant Cell Reports,2004,23:327-332.
72. Wu JiLin, Hao BingZhong. Wound-induced laticifer differentiation in Hevea brasiliensis shoots mediated by jas monic acid [J]. Journal of Rubber Research, 2002,5:53-63.
73. Yeang H Y, Cheong K F, Sunderasan E, et al. The 14.6kd rubber elongation factor (Hevb1) and 24kd (Hevb3) rubber particle proteins are recognized by IgE from patients with spinabifida and latex allergy [J]. The Journal of Allergy and Clinical Immunology,1996,98:628-639.
2.陈守才,邵寒霜,胡东琼,等.用RAPD技术鉴定橡胶树抗白粉病基因连锁标记[J].热带作物学报,1994,15(2)：21-26.
3.陈雄庭,王泽云,吴蝴蝶,张秀娟.橡胶树新种植材料-幼态自根无性系[J],热带作物学报,2002,23(1)：19-23.
4.陈雄庭.橡胶速生高产新型种植材料的培育与试种.2005,中国热带作物学会2005年学术(青年学术)研讨会
5.郝秉忠,吴继林.巴西橡胶树幼态无性系的高产特性[J].热带农业科学,1996,2：1-8.
6. 何波.橡胶树的幼态老态与全息胚理论[J].热带农业科学,1991,04：002.
7.何康,黄宗道.热带北缘橡胶栽培.[M].广州：广东科学技术出版社.1987
8.黄炎,郭庆水,徐立新,陈守才等.巴西橡胶树的分子生物学研究进展[J].安徽农业科学,2008,36(2)：442-445-
9.李守岭,庄南生.植物花药培养及其影响因素研究进展[J].亚热带植物科学,2006,35(3)：76-80
10.梁小莲,李辉亮,彭世清.巴西橡胶树HbTCTP基因的克隆及表达[J].分子植物育种,2009,7(1)：194-198.
11.刘松泉.巴西橡胶树幼态无性系的性质和利用[J].热带作物学术讨论会科技资料汇编,1979,13-20.
12.刘雷,尹钧,硫氧还蛋白的研究[J].东北农业大学学报,2003,34(2)：219-225.
13.鲁惠中,李辉亮,郭冬,彭世清.巴西橡胶树HbHDTl基因的克隆及表达分析[J].热带作物学报,2010,31(1)：59～64.
14.罗安定,陈守才,吴坤鑫等.AFLP在橡胶树优异种质研究中的应用[J].植物学报,2001,43(9)：941-947.
15.彭世清,吴坤金,陈守才.橡胶延长因子的cDNA的克隆和序列分析[J].华南热带农业大学学报,2000,6(2)：2-6
16.彭世清,傅湘辉,吴坤鑫,等.巴西橡胶树死皮病相关基因HbMyb1的结构分析及表达[J].植物生理与分子生物学学报,2003,29(2)：147-152.
17.潘衍庆主编.中国热带作物栽培学[M].北京：中国农业出版社,1998,3-5.
18.宿迁林业园林网.林木无性繁殖研究进展,http://lyj.suqian.gov.cn.
19.王颖,陈雄庭,张秀娟等.基因枪法将GAI基因导入巴西橡胶的研究[J].热带亚热带植物学报,2006,14(3)：179-182.
20.卫志明.花药和花粉培养.植物生理与分子生物学(第二版)[M].北京：科学出版社,1998,16-29.
21.许闻献.中国橡胶树割制改革30年[J].热带农业科学,2000,6：57-71.
22.杨立青-橡胶幼态的获得与利用[J].热带农业科技,2003,26(3)：16-18.
23.幼态课题组.幼态无性系在生产上的应用[J].热带农业科学,1994,2,1-3.
24.于俊红,黄绵佳,田维敏.巴西橡胶树橡胶生物合成调控的研究进展[J].安徽农学通报,2007,13(12)：38-40.
25.张治礼,郑学勤.树木发育阶段理论及其在橡胶树育种中的应用[J].热带亚热带植物学报,2001,9(3)：262-268.
26.中国橡胶网.中国成为越南天然橡胶主要出口国.http://www.cria.org.cn
27.中国橡胶市场网.中国橡胶工业协会会长范仁德讲话.2010,http://www.ex-rubber.com.
28.郑琼,马旭俊,杨传平.硫氧还蛋白(TRX)的研究进展[J].分子植物育种,2006,6(S)：78-82
29.曾日中,段翠芳,黎瑜,郝秉中.茉莉酸刺激的橡胶树胶乳cDNA消减文库的构建及其序列分析[J].热带作物学报,2003,(3)：34-37.
30. Arne Holmgren. Thioredoxin Catalyzes the Reduction of Insulin Disulfides by Dithiothreitol and Dihydrolipoamide[J].The journal of biological chemistry,1979, 254:9627-9632.
31. Arner E S J, Holmgren A. The thioredoxin system in cancer [J]. Seminars in Cancer Biology,2006,16:202-226.
32. Arokiaraj P, Jones H, Cheong K F, et al. Gene insertion into Hevea brasiliensis[J]. Plant Cell Reports,1994,13:425-426
33. Baker A, Payne C M, Briehl M M, et al. Thioredoxin,a gene found overexpressed in human cancer, inhibits apoptosis invitro and in vivo[J]. Cancer Res,1997,57:5162-5167.
34. Besse P, Lebrun P, Seguin M, et al. DNA fingerprints in Hevea brasiliensis using human minisatellite probes [J]. Heredity,1993,70:237-244.
35. Blomback M, Bjarke B,Herin P, et al. Neonatal aortic thrombosis.A possible clinical manifestation of congenital antithrombin III deficiency [J]. Acta Paediatr Scand,1977,63:297-301,1974
36. Broin M and Rey P. Potato plants lacking the CDSP32 plastidic thioredoxin exhibit overoxidation of the BAS12-cysteine peroxiredoxin and increased lipid Peroxidation in thylakoids under photooxidative stress [J]. Plant Physiol,2003,
132:1335-1343
37. Carvalho A P, Fernandes P A, Ramos M J. Similarities and differences in the thioredoxin superfamily[J].Progress in Biophysics and Molecular Biology,2006, 91:229-248.
38. Cornish K, Backhaus R A. Rubber transferase activity in rubber particles of guayule[J].Phytochemistry,1990,29:3809-3813
39. d'Auzac J, Jacob JL, Prevot JC, et al. The regulati on of cis-polyisoprene producti on (natural rubber) from Hevea brasiliensis [J]. Recent Res Plant Physiol, 1997,1:273-331
40. Deng X D, Fei X W, Huang J S. Isolation and analysis of rubber hevein gene and its promoter sequence [J]. Acta Botanica Sinica,2002,44:936-940.
41. Gelhaye E, Rouhier N, Navrot N, and Jacquot J P. The plant thioredoxin system [J]. Life Sci,2005,62:24-35
42. Goyvaerts E, Dennis M, Light D, et al. Cloning and sequencing of the cDNA encoding the rubber elongation factor of Hevea brasiliensis [J]. Plant Physiology, 1991,97:317-321
43. Hao Bingzhong, Wu Jilin. Laticifer differentiation in Hevea brasiliensis: Induced by exogenous jas monic acid and linolenic acid [J]. Annals of Botany, 2000,85:37-43
44. Holmgren A. Hydrogen donor system for Escherichia coli ribonucleoside-diphosphate reductase dependent upon glutathione [J]. Acad. Sci.,1976,73: 2275-2279.
45. Holmgren A. Thioredoxin:The amino acid sequence of the protein from Escherichia coli [J]. Biochem,1968,6:475-484.
46. Holmgren A. Thioredoxin and glutaredoxin systems [J]. Biol Chem,1989,264: 13963-13966.
47. Holmgren, A. Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide [J]. Biol. Chem,1979,254:9627-9632
48. Holmgren A, Bjornstedt M. Enzymatic reduction-oxidation of protein disulfides by thioredoxin [J]. Methods Enzymol,1984,107:295-300.
49. Holmgren J, Lindblad H, Fredman P, et al. Comparison of receptors for cholera and Escherichia coli enterotoxins in human intestine [J]. Gastroenterology,1985, 89:27-35.
50. Holmgren C, Johansson C, Berndt M E, et al. Thioredox control via thioredoxin
and glutaredoxin systems [J]. Cellular Information Processing,2005,1375-1377.
51. Jaglo K R, Gilmours J, Zarka D G, et al. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing to lerance [J]. Science,1998,280: 104-106.
52. Javier Andres Juarez-Dlaz, Bruce McClure, Sonia Vazquez-Santana. A Novel Thioredoxin h Is Secreted in Nicotiana alata and Reduces S-RNase in Vitro [J]. The journal of biological chemistry,2006,281:3418-3424.
53. Juttner J, Olde D, Langridge P, et al. Cloning and expression of a distinct subclass of plant thioredoxins [J]. European Journal of Biochemistry,2000,267: 7109-7117.
54. Kim Y B, Garbisu C, Pickering L J, et al. Thioredoxinh overexpressed in barley seeds enhances selenite resistance and uptake during germination and early seedling development [J]. Planta,2003,218:186-191.
55. Laurent T C, Moore E C, Reichard P, et al. Enzymatic synthesis of deoxyribonucleotides.Ⅳ.Isolation and characterization of thioredoxin, the hydrogen donor from Escherichia coli [J]. Biol.Chem,1964,239:3436-3444.
56. Lee K K, Murakawa M, Takahashi S et al. Purification, molecule cloning, and characterization of TRX32, a novel thioredoxin-related mammalian protein of 32 kD [J]. Biol Chem,1998,273:19160-19166.
57. Light D R, Lazarus R A, Dennis M S et al. Rubber elongation by farnesyl pyrophosphate synthases involves a novel switch in enzymes tereospecificity [J]. The Journal of Biological Chemistry,1989,264:18598-18607.
58. Low F C, Safiah A, Hafsah J, et al. Recent advance in the development of molecular markers for Hevea brasiliensis [J].Journal of Natural Rubber Research, 1996,11:32-44.
59. Mannervik B, Guthenberg C, Jensson H, et al. Isozymes of glutathione S-transferases in rat and human tissues, in Functions of Glutathione:Biochemical, Physiological, Toxicological, and Clinical Aspects [J]. Larsson A, Orrenius S, Holmgren A, and Mannervik B eds,1983,1:75-88.
60. Martin J L. Thioredoxin2a fold for all reasons [J].Structure,1995,3:2452250.
61. Miao Z H, John J G Molecularcloning, characterization and expression of Mn superoxide dismutase from the rubber tree(Hevea brasiliensis) [J]. Plant Molecular Biology,1993,23:267-277.
62. Okada K, Sato T, Nakagawa M, et al. Five geranylgeranyldiphosphate synthases expressed in different organs are localized into three subcellular compartments in Arabidopsis [J]. Plant Physiology,2000,122:1045-1056.
63. Roland Cazalis, Pablo Pulido, Thierry Aussenac, et al. Cloning and characterization of three thioredoxin h isoforms from wheat showing differential expression in seeds [J]. Journal of Experimental Botany,2006,57:2165-2172.
64. Sano H, Sata T, Nanri H, et al. Thioredoxin is associ2 ated with endotoxin tolerance in mice [J]. Crit Care Med,2002,30:192-194.
65. Sederberg A, Sahaf B, Rosn A et al. Thioredoxin reductase, a redox active selenoprotein, is secreted by normal and neoplastic cells:presence in human plasma [J]. Cancer Res,2000,60:2281-2289.
66. Southorn W A. Physiology of Hevea latex flow [J]. Rubber Res,1969,21: 494-512.
67. Southorn W A, Yip E. Latex flow studies III Electrostatic considerations in colloidal stability of fresh Hevea latex [J]. Rubber Res,1968,20:210-215.
68. Tang C, Qi J, Li H, et al. A convenient and efficient protocol for isolating high-quality RNA from latex of Hevea brasiliensis (para rubber tree) [J]. Biochem Biophys Methods,2007,70:749～754.
69. Tian WeiMin, Shi MinJing, Wu JiLin, et al. Localized Effects of mechanical wounding an exogenous jasmonic acid on the induction of secondary laticifer differentiati on in relation to the distribution of jasmonic acid in Hevea brasiliensis[J].Acta Botanica Sinica,2003,45:1366-1372.
70. Ueno M, Masutani H, Arai R J, et al. Thioredoxin dependent redox regulation of p532mediated p21 activation [J]. Biol Chem,2000,274:35809-35815.
71. Venkata C P, Priya P, Sarawathy A C, et al. Identification, cloning and sequence analysis of a dwarf genome specific RAPD marker in rubber tree [J]. Plant Cell Reports,2004,23:327-332.
72. Wu JiLin, Hao BingZhong. Wound-induced laticifer differentiation in Hevea brasiliensis shoots mediated by jas monic acid [J]. Journal of Rubber Research, 2002,5:53-63.
73. Yeang H Y, Cheong K F, Sunderasan E, et al. The 14.6kd rubber elongation factor (Hevb1) and 24kd (Hevb3) rubber particle proteins are recognized by IgE from patients with spinabifida and latex allergy [J]. The Journal of Allergy and Clinical Immunology,1996,98:628-639.