摘要
番木瓜是番木瓜科(Caricaceae)番木瓜属(Carica L.)植物,是热带、亚热带重要果树之一。番木瓜果实属呼吸跃变型,在18℃以上,采后果实迅速成熟、软化。番木瓜对低温敏感,不能冷藏运输,因此在运输和储藏中的损失是巨大的,高达23.7%。近年来,随着分子生物学的发展,延缓果实衰老的研究转向采用基因工程培育耐储运新品种上。番木瓜果实成熟软化相关基因克隆及其遗传转化的研究比较少,没有见到耐储运新品种商业化生产的报道。为了解番木瓜果实成熟的分子机理,挖掘更多更有效的果实抗软化基因,以番木瓜不同成熟度果实为试材,采用cDNA-AFLP技术分析了果实成熟基因差异表达情况,并利用RACE技术克隆了差异表达基因。果胶裂解酶(Pectate lyase, PL)与β-半乳糖苷酶(β-Galactosidase,β-Gal)都参与了果胶物质的降解过程,与果实成熟软化密切相关。克隆了番木瓜果胶裂解酶基因,构建了PL反义植物表达载体以及PL与β-Gal双价反义植物表达载体,建立了体胚发生系统,并通过农杆菌介导法将两个载体分别转化番木瓜,获得了抗性体胚。本研究的主要结果如下:
1番木瓜不同成熟度果实cDNA-AFLP分析
利用cDNA-AFLP技术对破色期和半黄期番木瓜果实进行基因差异表达分析,获得了50个T DFs,其中28个与Genbank数据库中的功能基因同源,5个与未知功能的基因同源,17个未找到同源基因。经生物信息学分析,这28个与已知功能基因同源的TDFs分别参与了果实成熟过程中基因表达调控、DNA与蛋白质合成及运输、蛋白质降解、能量代谢、营养物质代谢和植物逆境胁迫反应的过程。11个与基因表达调控有关的差异片段中,7个与信号转导有关,3个为转录因子,1个与转录后调控有关。
2番木瓜果实成熟差异表达基因及Actin基因的克隆与分析
采用cDNA末端快速扩增(RACE)技术克隆了5个果实成熟差异表达基因和一个肌动蛋白基因,并用半定量RT-PCR分析了它们在不同成熟度番木瓜果实中的表达量差异。
克隆了番木瓜过氧化物酶基因,CpPOD全长cDNA为1124 bp。序列分析发现,没有起始密码子,可能是测序错误或碱基缺失造成的。不同成熟度CpPOD基因表达量分析表明,绿色期表达量很低,破色期表达量非常大,半黄和全黄时表达量有所下降。
克隆了番木瓜MYB转录因子,CpMYB全长cDNA为1507 bp,含有一个879 bp的开放阅读框,编码292个氨基酸。基因表达分析表明,CpMYB基因随着番木瓜成熟表达量逐渐增加,衰老期又开始降低。
克隆了番木瓜GDP-D-甘露糖焦磷酸化酶基因,CpGMP全长cDNA为1544 bp。以基因组DNA为模板扩增ORF长1775 bp,含有4个外显子,3个内含子;cDNA为模板扩增ORF长1096 bp,编码361个氨基酸。半定量RT-PCR分析结果表明,CpGMP基因随着番木瓜果实成熟表达量逐渐增加,软化时又逐渐降低。
克隆了番木瓜20S蛋白酶体α亚基6蛋白基因,CpPAA1全长cDNA为1025 bp,含有一个741 bp的开放阅读框,编码246个氨基酸。表达量分析发现,CpPAA1基因随着番木瓜成熟表达量逐渐增加,衰老期又开始降低。
克隆了番木瓜植物类受体蛋白激酶基因3'端序列,cDNA长924 bp。表达量分析表明,绿色期表达量最高,然后随着果实成熟和衰老表达量逐渐降低。
克隆了番木瓜肌动蛋白基因,CpActin全长cDNA为1533 bp,含有一个1134 bp的开放阅读框,编码377个氨基酸。
3番木瓜果实果胶裂解酶基因克隆及反义植物表达载体的构建
采用cDNA末端快速扩增方法,获得了番木瓜果实果胶裂解酶基因的完整3'端和部分5'端序列。然后,根据本实验室已经获得的PL基因5'端DNA序列及作者得到的3'端序列设计上、下游引物,以番木瓜基因组DNA为模板扩增得到了PL基因的开放阅读框。该序列长1464 bp,含有4个外显子,3个内含子,编码385个氨基酸。然后构建了PL基因反义植物表达载体pBP,并将其导入根癌农杆菌EHA105中。
4番木瓜PL和β-Gal基因的双价植物反义表达载体的构建
在番木瓜PL和β-Gal基因序列的基础上,设计特异引物,分别扩增保守区序列,将其分别反向插入植物表达载体pCAMBIA1301-35S-GUS-Nos的CaMV 35S启动子和Nos终止子之间,构建了反义表达载体pCP和pCG。然后用两种不同的方法将带有完整启动子和终止子的PL和β-Gal基因引入pCAMBIA2301中,获得了PL和β-Gal的双价植物反义表达载体pCPG,并对两种方法进行了比较。
5番木瓜体胚发生系统的建立及遗传转化
以番木瓜幼胚为外植体,研究不同激素配比对胚性愈伤组织及体胚诱导的影响。结果表明,胚性愈伤组织诱导的最适培养基为改良MS+10 mg/L 2,4-D +2 mg/L KT +0.5 mg/L BA +30 g/L蔗糖+400 mg/L谷氨酰胺,体胚诱导最适培养基为改良MS+10 mg/L 2,4-D +2 mg/L KT+30 g/L蔗糖+400 mg/L谷氨酰胺。利用农杆菌介导法将构建好的PL基因反义表达载体和PL与β-Gal基因双价反义表达载体分别转化番木瓜,获得了抗性体胚。
Papaya, Carica papaya, a member of the family Caricaceae, is one of the most important tropical and subtropical fruit trees. Like most climacteric fruit, papaya ripens quickly after harvest and then soften and deteriorate rapidly. It is also sensitive to low temperature and cannot be stored in cool conditions. Hence, large losses during the storage and transportation of papaya are caused. Up to 23.7% of the harvest is lost in transit from the farmer to the consumer. With the molecular biology development, genetic engineering was used to modify genes in order to regulate fruit ripening. Studies of cloning and transformation of fruit ripening-related genes were rare and the reports of genetically modified varieties on sale were not seen. In order to know more about molecular mechanism of fruit ripening and to seek more effective anti-softening genes, cDNA-AFLP technique was used to identify differential gene expression during papaya fruit ripening and some of the differential expression genes were cloned by RACE. Pectate lyase gene was cloned too. PL antisense expression vector and PL andβ-Gal antisense expression vector were constructed and transformed into papaya mediated by Agrobacterium tumefaciens co-transformation, and the kan-resistant somatic embryo were obtained. The major results obtained from the study are summarized as follow:
1 Studies on differential gene expression of papaya fruit at different ripening stages by means of cDNA-AFLP technique
Fifty TDFs were obtained from the analysis of color break and half yellow stage of papaya fruit by cDNA-AFLP. Bioinformatics analysis showed that twenty eight TDFs were homologous to known function genes, five TDFs were homologous to uncharacterized genes, while seventeen TDFs did not show significant matches to any genes in the Genbank database. Twenty eight genes of known function TDFs could be divided into different functional groups including gene expression regulation, DNA and protein synthesis and transport, protein degradation, energy metabolism, nutrient metabolism and stress responding. Among the eleven expression regulation genes, seven was signal transduction genes, three was transcription factors, and one was post-transcriptional regulation gene. These results indicated that fruit ripening and senescence is a complicate physiological and biochemical processes and should be regulated by a complex system of signal induction and physi-biochemical metablism.
2 Cloning and analysis of differential expression of fruit ripening genes and Actin gene from papaya
Five differential expression genes and one Actin gene were cloned by rapid amplification of cDNA ends (RACE) from papaya. The gene expression patterns in papaya fruit at different ripening stages was analyzed by semi-quantitative RT-PCR.
The full length cDNA of CpPOD was 1124 bp. Sequence analysis showed that the start codon was could not find, this may be caused by mistaken sequencing or base losting. Semi-quantitative RT-PCR analysis showed that the expression of CpPOD was highest in color break stage and decreased when fruit began to ripen and soften.
A full-length cDNA clone encoding MYB was cloned from papaya. The full length cDNA of CpMYB was 1124 bp with an opening reading frame of 879 bp encoding a protein with 292 amino acids. Semi-quantitative RT-PCR analysis showed that the expression of CpMYB was increased gradually with papaya fruit maturing and decreased when fruit began to senesce.
GDP-mannose pyrophosphorylase gene was cloned from papaya. The full length cDNA of CpGMP was 1544 bp. Open reading frame which encoded 361 amino acids was cloned from genome DNA and cDNA. ORF of DNA sequence was 1775 bp which including 4 extrons and 3 introns. Semi-quantitative RT-PCR analysis showed that the expression of CpGMP increased gradually with papaya fruit maturing and decreased when fruit began to soften.
Proteasome subunit alpha type-6 gene was cloned from papaya. The full length cDNA of CpPAA1 was 1025 bp with an ORF of 741 bp encoding a protein with 246 amino acids. Semi-quantitative RT-PCR analysis showed that the expression of CpPAA1 increased gradually with papaya fruit maturing and decreased when fruit began to senesce.
CpSRF3 partial sequence was obtained by 3'RACE which encoded 128 amino acids. Gene domain analysis showed that CpSRF3 is a kind of receptor-like protein kinases with a leucine-rich repeat. Semi-quantitative RT-PCR analysis showed that the expression of CpSRF3 decreased gradually with papaya fruit ripening and senescence. A full-length cDNA clone encoding Actin was amplified from papaya. Sequence analysis showed that it contained a complete ORF of 1134 bp coding for 377 amino acids. Actin gene was often used as an internal standard in gene expression analysis.
3 Cloning of pectate lyase gene from papaya and construction of antisense expression vector
Complete 3' and part 5' terminal sequence of PL gene were obtained by RACE from papaya. Using two specific primers, open reading frame of 1464 bp encoding 385 amino acids was cloned from genomic DNA. There were 4 extrons and 3 introns in the sequence. Then, antisense expression vector pBP was constructed and transformed into Agrobactrium tumefaciens EHA105.
4 Construction of PL andβ-Gal antisense expression vector of papaya
PL andβ-Gal conserved domains were cloned using specific primers designed according to those gene sequences. The PL andβ-Gal cDNA were inserted into the site between CaMV 35S promoter and Nos terminator of the expression vector pCAMBIA1301-35S-GUS-Nos with reverse orientation respectively. So, two antisense expression vectors pCP and pCG were obtained. Then, PL andβ-Gal antisense genes with 35S promoter and Nos terminator were inserted into expression vector pCAMBIA2301 together to generate double gene plant expression vector pCPG by different mode.
5 Studies of establishment of somatic embryogenesis and transformation system of papaya
The effects of different hormones on the somatic embryogenesis from papaya immature zygotic embryo were studied. Results showed that the appropriate medium for callus induction was modified MS media supplemented with 10 mg/L 2,4-D and 2 mg/L KT and 0.5 mg/L BA and 30 g/L sucrose and 400 mg/L glutamine, the appropriate medium for somatic embryogenesis was modified MS media supplemented with 10 mg/L 2,4-D and 2 mg/L KT and 30 g/L sucrose and 400 mg/L glutamine. PL antisense expression vector and PL,β-Gal antisense expression vector were transformed into papaya mediated by Agrobacterium tumefaciens co-transformation, and the kan-resistant somatic embryo were obtained.
引文
[1]安华明,陈力耕,樊卫国,等.高等植物中维生素C的功能、合成及代谢研究进展[J].植物学通报, 2004, 21(5): 608-617.
[2]卞伟华,江昌俊,王朝霞.桃叶片β-半乳糖苷酶基因全长cDNA克隆及原核表达[J].园艺学报, 2006, 33 (4): 721~724.
[3]蔡盛华,陆修闽,黄雄峰,等.番木瓜的保健药用与庭院栽培要点[J].福建果树, 2003, (2): 10-11.
[4]柴明良,金斗焕.农杆菌介导的蝴蝶兰基因转化系统的建立[J].园艺学报, 2004, 31(4): 537-539.
[5]陈观水,潘大仁,周以飞,等.甘薯NPR1基因半定量RT-PCR检测方法的建立[J].福建农林大学学报(自然科学版), 2007, 36(1): 56-59.
[6]陈惠萍,陈晓敏,蔡世英.香蕉果实后熟过程中活性氧代谢的变化[J].园艺学报, 2000, 27(5): 369-370.
[7]陈俊,王宗阳.植物MYB类转录因子研究进展[J].植物生理与分子生物学学报, 2002, 28(2): 81-88.
[8]陈昆松,徐昌杰,张上隆.富含多糖猕猴桃果实组织中总RNA提取方法的改进[J].植物生理学通讯, 1998, 34(5): 371-373.
[9]陈璇,李文正,邵岩,等.动植物中RNA结合蛋白的研究进展[J].生物技术通报, 2007, 3: 9-15.
[10]陈中海.马蹄莲、石斛和姜凝集素基因的克隆及分析[D].复旦大学博士论文, 2004.
[11]程杰山,沈火林,朱鑫,等.果实成熟过程中细胞壁多糖的变化.北方园艺, 2006, 5: 70-72.
[12]戴顺洪,李良材,丁月云,等.水稻基因枪法多基因转化研究[J].遗传学报, 1998, 25(4): 345-350.
[13]邓雪柯,殷建华,曹毅. 3种5'RACE技术的比较与优化[J].成都医学院学报, 2007, 2(1): 20-25.
[14]丁勇,陈庆波,徐春雷,等.油菜油体钙蛋白基因BnClo1的克隆和表达[J].作物学报, 2008, 34(11): 1921-1928.
[15]段学武,张昭其,季作梁. PG酶与果实的成熟软化[J].果树学报, 2001, 18(4): 229-233.
[16]傅荣昭,孙勇如,贾士荣.植物遗传转化技术手册[M].北京:中国科学技术出版社, 1994: 141-144.
[17]龚亚明,毛伟华.果实成熟分子生理的研究进展[J].浙江农业学报, 2002, 14(4): 244-248.
[18]郭德章,鄢铮.番木瓜体细胞胚胎发育与植株再生[J].亚热带植物通讯, 2000, 29(1): 38-40.
[19]郭丽琼,林俊芳,杨丽卿,等.应用cDNA-AFLP技术分离草菇冷诱导表达基因[J].园艺学报, 2005, 32(1): 54-59.
[20]郭晓强.线粒体蛋白质运输机制[J].生物学杂志, 2005, 22(3): 5-7.
[21]韩继成.植物乙烯受体及转基因育种研究进展[J].分子植物育种, 2004, 2(2): 157-163.
[22]韩继成,方宣钧.编码荔枝乙烯受体(LcERS1)的cDNA基因的克隆与分析[J].分子植物育种, 2003, 1(3): 351-356.
[23]郝艳琴,梁爱华,柴宝峰.蛋白质合成肽链释放因子的多功能性[J].生命的化学, 2007, 27(5): 386-389.
[24]何琳,朱本忠,罗云波.转反义LeEIL2基因番茄果实采后部分生理特性[J].中国农业大学学报, 2006, 11(1): 57-60.
[25]何玮毅,陈晓静,申艳红,等.番木瓜叶片、叶柄胚性愈伤组织的诱导及其体胚植株的发生[J].热带作物学报, 2008, 29(6): 690-697.
[26]胡英考,李雅轩,蔡民华,等.提高植物维生素含量的基因工程[J].中国生物工程杂志, 2004, 24(5): 20-23.
[27]华南农业大学主编.果树栽培学各论(南方本) [M].中国农业出版社, 1989: 243.
[28]黄俊潮,叶克难,陈谷,等.番木瓜体细胞胚发生及发育的影响因素[J].中山大学学报(自然科学版),1996, 35(4): 90-94.
[29]黄文敏,邢伟,李敦海,等.外源抗坏血酸对烟草细胞生长及衰老的影响[J].农业环境科学学报, 2006, 25(5): 1157-1161.
[30]姜立杰,张开春,张晓明. cDNA-AFLP技术及其在基因表达研究中的应用[J].中国生物工程杂志, 2003, 23(12): 82-86.
[31]金昌海,水野雅史,汪志君,等.苹果β-半乳糖苷酶对细胞壁多糖降解特性的研究[J].扬州大学学报(农业与生命科学版), 2002, 23 (4): 71-74.
[32]金基石,李永红,单宁伟,等.抗坏血酸提高月季切花失水胁迫耐性与增加APX活性的关系[J].园艺学报, 2006, 33(2): 333-337.
[33]鞠戎,田颖川,沈全光,等.番茄多聚半乳糖醛酸酶cDNA的克隆及其对番茄中PG表达的反义抑制[J].生物工程学报, 1994, 10(2): 96-102.
[34]寇晓虹,罗云波,田慧琴,等.多聚半乳糖醛酸酶(PG)反义基因转化加工番茄[J].食品科学, 2007, 28(3): 187-191.
[35]冷春旭.陆地棉TM-1再生体系的建立及其胚胎发生mRNA差异显示分析[J].中国农业科学院硕士学位论文, 2005.
[36]李大志,陈霄,邓子牛,等.应用cDNA-AFLP技术分离应答BABA诱导的番茄抗病相关基因[J].湖南农业大学学报(自然科学版), 2007, 33(1): 32-36.
[37]李宏,王新力.植物组织RNA提取的难点及对策[J].生物技术通报, 1999, 15(1): 36-39.
[38]李红双,崔德才.利用多聚半乳糖醛酸酶反义基因转化选育耐贮中国樱桃[J].生物技术通讯, 2006, 17(6): 885-887.
[39]李惠,肖璐,祝建波,等.加工番茄多聚半乳糖醛酸酶(PG)基因的cDNA克隆和全序列测定[J].生物技术, 2001, 11(2): 1-4.
[40]李松,陈丽新,谭芳,等.农杆菌介导及基因枪转化在我国甘蔗遗传转化的应用研究现状[J].2004, 5: 1-5.
[41]李天然,马庆虎.番茄ACC合成酶反义基因对河套蜜瓜的转化[J].植物学报, 1999, 41(2): 142-145.
[42]李小平,马媛媛,李鹏丽,等.利用RNA干扰技术敲减rlpk2基因的表达可以延缓大豆叶片衰老[J].科学通报, 2005, 50(11): 1090-1096.
[43]李亚新.首例商品化的转基因果树-番木瓜[J].园艺学报, 2000, 28(1): 51.
[44]李越. 20S蛋白酶体在人视网膜色素上皮细胞老化中的改变及其在诱导衰老中的作用[D].第四军医大学博士学位论文, 2008: 28-29.
[45]林莹.番木瓜果胶裂解酶基因的克隆.福建农林大学硕士论文[D], 2006, 37-39.
[46]刘志勇,杜永臣,王孝宣,等.高温胁迫下番茄叶片差异表达基因的cDNA-AFLP分析[J].园艺学报, 2008, 35 (7): 1011-1016.
[47]卢钢,曹家树. cDNA-AFLP技术在植物表达分析上的应用[J].植物学通报, 2002, 19(1): 103-108.
[48]陆璐,王鸣,郑学勤,等.哈蜜瓜ACC氧化酶cDNA克隆及其序列分析[J].园艺学报, 2000, 27(1): 32-36.
[49]路梅,徐传雨,郭卫东.植物LRR类受体蛋白激酶的研究进展[J].浙江师范大学学报(自然科学版), 2006, 29(3): 322-325.
[50]吕平,韦丽君,苏文潘,等.番木瓜组织培养概况[J].热带农业科技, 2006, 29(2): 31-34.
[51]卢圣栋.现代分子生物学实验技术[M].北京:高等教育出版社, 1993: 50.
[52]陆胜明,金勇丰,张耀洲,等.果实成熟过程中细胞壁组成的变化[J].植物生理学通讯, 2001, 3(37): 246-249.
[53]马春花,马锋旺,李明军,等.不同叶龄苹果叶片抗坏血酸含量与其代谢相关酶活性的比较[J].园艺学报, 2007, 34(4): 995-998.
[54]马庆虎,王莉梅,宋艳茹,等.肥城桃中多聚半乳糖醛酸酶基因的分离及其表达研究[J].植物学报, 1999, 41(3): 263-267.
[55]缪晏珉,李娜,任旭琴,等.一个辣椒肌醇半乳糖苷合成酶同源基因全长cDNA的克隆与低温表达分析[J].园艺学报, 2008, 35(11): 1671-1675.
[56]倪贺,李海航,黄文芳,等.核糖核苷酸还原酶研究[J].科技导报, 2008, 26(8): 79-83.
[57]倪晓光,赵平.泛素-蛋白酶体途径的组成和功能[J].生理科学进展, 2006, 37(3): 255-258.
[58]潘瑞炽主编.植物生理学[M].北京:高等教育出版社, 2001.
[59]乔玉山,宋长年,王新卫,等.砂梨ACC合酶cDNA全长克隆及其反义与正义表达载体构建[J].南京农业大学学报, 2008, 31(4): 25-31.
[60]邱为民,张思仲,武辉,等.一种新的cDNA末端快速扩增获取全长cDNA的方法[J].遗传, 2001, 23(5): 480-482.
[61]曲泽洲,李三凯,武元苏,等.枣贮藏保鲜技术研究[J].中国农业科学, 1987, 20(2): 86-91.
[62]饶雪琴,李华平.番木瓜美中红品种体胚转化体系的建立[J].华中农业大学学报, 2007, 26(3): 293-296.
[63]饶雪琴,李华平.红肉小果型番木瓜品种“美中红”体胚的诱导[J].植物生理学通讯, 2007, 43(1): 73-76.
[64]生吉萍,申琳,罗云波.果实成熟衰老相关酶的研究进展[J].食品与机械, 2000, 77(3): 7-9.
[65]石晶盈,刘爱媛,冯淑杰,等.番木瓜内生细菌MG-Y2的鉴定及其生防作用[J].果树学报, 2007, 24(6): 810-814.
[66]石琰璟.利用SMART RACE克隆STGA3 cDNA的5'末端[J].青岛科技大学学报, 2006, 27(1): 16-19.
[67]宋爽,赵宏巍,曹俊然,等.两个大豆类受体蛋白激酶基因的克隆及其结构和功能的初步分析[J].植物生理与分子生物学学报, 2002, 28(3): 241-246.
[68]宋素胜,谢道昕.泛素蛋白酶体途径及其对植物生长发育的调控.植物学通报, 2006, 23(5): 564-577.
[69]孙保娟,曹家树,黄细松,等.白菜离体春化相关基因表达的cDNA-AFLP分析[J].园艺学报, 2006, 33 (6): 1341-1344.
[70]汤福强,刘愚. ACC合成酶基因及其反义基因转化番茄获得转基因植株[J].科学通报, 1994, 39(24): 2280-2283.
[71]王春霞,简志英. ACC合成酶基因及反义基因对西瓜的遗传转化[J].植物学报, 1997, 39 (5): 445-450.
[72]王关林,方宏筠.植物基因工程(第二版)[M].北京:科学出版社, 2005: 755.
[73]王贵禧,韩雅珊.猕猴桃果实中PME、PG及其抑制因子的研究[J].中国农业大学学报, 1998, 3(1): 88-94.
[74]王晓飞.若干核果类果树及番木瓜β-半乳糖苷酶同源基因的克隆[D].福建农林大学硕士学位论文, 2005.
[75]韦平和,吴滔,吴梧桐,等.低特异性L-苏氨酸醛缩酶基因工程菌的构建[J].药物生物技术, 2000, 7(2): 86-89.
[76]魏绍冲,陈昆松.成熟猕猴桃果实中乙烯受体基因Ad-ETR2的克隆与序列分析[J].果树学报, 2004, 21(6): 544-547.
[77]魏绍冲,陈汝,赵相娟,等.冬枣两个ACC氧化酶基因的cDNA克隆及其表达模式[J].园艺学报, 2008, 35(5): 643-648.
[78]魏绍冲,彭福田,束怀瑞,等.冬枣两个乙烯受体编码基因的克隆及序列分析[J].园艺学报, 2007, 34(2): 333-338.
[79]吴显荣.番木瓜的生物化学[J].热带作物科技, 1992, 1: 5-11.
[80]夏杏洲,胡雪琼.番木瓜资源的开发利用与产业化发展.热带农业科学, 2002, 22(4): 72-77.
[81]萧介夫,杨居成,余聪安,等. http://www.sinica.edu.tw/~npagrbt/mpage2/mpage2-4/mpage2-4-35.htm, 2001.
[82]肖月华,罗明,韦宇拓,等.棉花纤维起始期基因表达的cDNA-AFLP分析[J].农业生物技术学报, 2003, 11(1): 20-24.
[83]谢宏峰,曹银生,薛仁镐.影响复合针介导的大豆遗传转化效率的主要因素研究[J].生物技术通讯, 2007, 18(6): 946-948.
[84]谢庆昌.采收成熟度、贮藏温度、后熟温度及强制热风处理对“台农二号”番木瓜品质的影响[J ].中国园艺, 2001, 47(4): 391-408.
[85]熊爱生,姚泉洪,李贤,等. ACC氧化酶和ACC合成酶反义RNA融合基因导入番茄和乙烯合成的抑制[J].实验生物学报, 2003, 36(6): 428-434.
[86]徐金会.漫谈蛋白酶复合体[J].生物学教学, 2005, 30(9): 2-3.
[87]杨培生,钟思现,杜中军,等.我国番木瓜产业发展现状和主要问题[J].中国热带农业, 2007, 4: 8-10.
[88]杨奇志,赵琦,孔建强.基因枪技术在农作物基因转化中的应用和进展[J].生物技术通报, 2003, 6: 14-23.
[89]姚玉新,赵玲玲,郝玉金,等.改良热硼酸法高效提取苹果果实RNA[J].果树学报, 2005, 22(6): 737-740.
[90]叶克难,马蕾,李宝健.番木瓜悬浮培养的体胚发生和植株再生[J].植物学报, 1991, 33: 564-568.
[91]于湄,叶长明,李宝健. PRV外壳蛋白基因转化番木瓜的研究[J].佛山科学技术学院学报(自然科学版), 2001, 19(1): 59-61.
[92]余叔文,汤章城.植物生理与分子生物学(第二版)[M].北京:科学出版社, 1998.
[93]于娅,刘传亮,马峙英,等.基因枪转化技术在棉花遗传转化上的应用[J].棉花学报, 2003, 15(4): 243-247.
[94]曾继吾,易干军,张秋明.番木瓜体胚发生及植株再生研究[J].果树学报, 2003, 20(6): 471-474.
[95]张德有,徐碧玉,金志强.香蕉果胶裂解酶基因的克隆[J].植物生理与分子生物学学报, 2003, 29(5): 401-404.
[96]张丽,哈斯阿古拉,张竞秋,等.甜瓜多聚半乳糖醛酸酶基因cDNA的克隆和序列分析[J].内蒙古大学学报(自然科学版), 2003, 34(3): 304-307.
[97]张嵩,张光伦,曾秀丽.纤维素酶和多聚半乳糖醛酸酶与果实成熟[J].果树学报, 2005, 22(5): 532-536.
[98]张艳君,朱志峰,陆融,等.基因表达转录分析中内参基因的选择[J].生物化学与生物物理进展, 2007, 34(5): 546-550.
[99]赵志英,周鹏,曾宪松,等.核酶基因转化番木瓜的研究[J].热带作物学报, 1998, 19(2): 20-25
[100]赵云峰,林河通,林娇芬,等.果实软化的细胞壁降解酶及其调控研究进展[J].仲恺农业技术学院学报, 2006, 19(1): 65-70.
[101]郑军荣,周鹏,洪葵. phaC1、phaC2基因双价植物表达载体的构建及其转基因烟草的获得[J].分子植物育种, 2003, 1(1): 58-65.
[102]周鹏,郑学勤.根癌农杆菌介导的环斑病毒外壳蛋白基因转化番木瓜的研究[J].热带作物学报, 1993, 14(2): 71-79
[103]周鹏,郑学勤. PRV-CP-SN转基因番木瓜表达与抗病能力的研究[J].热带作物学报, 1996, 17(2): 84-87.
[104]周胜军.园艺作物成熟和衰老的分子生物学[J].生物学杂志, 2002, 19(5): 5-8.
[105]朱海生,潘东明,温庆放,等.反义乙烯受体FaEtr2基因对草莓的转化[J].园艺学报, 2008, 35(11): 1587-1594.
[106]朱海生,温庆放,林义章,等.草莓乙烯受体FaEtr1和FaErs1基因的克隆及其反义表达载体的构建[J].农业生物技术学报, 2007, 15(4): 684-688.
[107]庄军平,苏菁,陈维信.香蕉果实β-半乳糖苷酶基因cDNA克隆及序列分析[J].西北植物学报, 2006, 26(1): 0018-0022.
[108]邹韵霞,郭惠.番木瓜胚珠高频率体胚发生和植株再生[J].中山大学学报(自然科学版), 1992, 31(3): 60-65.
[109] Asif M H, Dhawan P, Nath P. A simple procedure for the isolation of high quality RNA from ripening banana fruit[J]. Plant Molecular Biology reporter, 2000, 18: 109-115.
[110] Awasthi N, Wagner B J. Suppression of human lens epithelial cell proliferation by proteasome inhibition, a potential defense against posterior capsular opacification[J]. Investigative Ophthalmology and Visual Science, 2006, 47(10): 4482-4489.
[111] Ayub R, Guis M, Amor M B, et al. Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits[J]. Nat Biotechnol, 1996, 14 (7): 862-866.
[112] Bachem C W B, Horvath B, Trindade L, et al. A potato tuber-expressed mRNA with homology to steroid dehydrogenases affects gibberellin levels and plant development[J]. Plant Journal, 2001, 25(6): 595-604.
[113] Bachem C W B, Oomen R J F J, Visser R G F. Transcript imaging with cDNA-AFLP: a step-by-step protocol[J]. Plant Mol Biol Rep, 1998, 16:157-173.
[114] Bachem C W B, van der Hoeven R S, de Bruijn S M, et al. Visualization of diferential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development[J], Plant Journal, 1996, 9(5): 745-753.
[115] Bahrami A R, Gray J E. Expression of a proteasome alpha-type subunit gene during tobacco development and senescence[J]. Plant Mol Biol. 1999, 39(2): 325-333.
[116] Balde A, Gouveia M M C, Pais M S. Papaya (Carica papaya) fruit ripening I- Pectinmethylesterase (PME) cDNA cloning and expression during fruit development and ripening[J]. Nato Science Series Sub Series I Life and Behavioural Sciences, 2005, 368: 224-230.
[117] Barth C, Moeder W, Klessig D F, et al. The timing of senescence and response to pathogens is altered in the ascorbate-deficient Arabidopsis mutant vitamin c-1[J]. Plant Physiology. 2004, 134(4): 1784-1792.
[118] Bassett C L, Artlip T S. Isolation of an ETR1 ethylene receptor homologue from peach (Prunus persica (L.) Batsch) [J]. HortScience, 1999, 34: 542.
[119] Bendtsen J D, Nielsen H, von Heijne G, et al. Improved prediction of signal peptides: SignalP 3.0[J]. J Mol Biol, 2004, 340: 783-795.
[120] Benjamin L.基因VIII[M].北京:科学出版社, 2005: 104.
[121] Bilaud T, Koering C E, Binet-Brasselet E, et al. The telobox, a myb-related telomeric DNA binding motif found in proteins from yeast, plants and human[J]. Nucleic Acids Research, 1996, 24(7): 1294-1303.
[122] Bonghi C, Ferrarese L. Endo-b-1,4-glucanases are involved in peach fruit growth and ripening, and regulated by ethylene[J]. Physiologia Plantarum, 1998, 102: 346-352.
[123] Borraccino G, Mastopasqua I, De L S, et al. The role of ascorbic acid system in delaying the senescence of oat (Avena sativa L.) leaf segments[J]. Journal of Plant Physiology, 1994, 114: 161-166.
[124] Brummell D A, Hall B D, Bennett A B. Antisense suppression of tomato endo-1,4-beta- glucanase Ce12 mRNA accumulation increases the force required to break fruit abscission zones but does not affect fruit softening[J]. Plant Mol Biol, 2000, 40: 615 - 622.
[125] Carpita N C, Gibeaut D M. Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth[J]. Plant Journal, 1993, 3: 1-30.
[126] Chen Y T, Lee Y R, Yang C Y, et al. Factors affecting the expression patterns of two papaya 1-aminocyclopropane-1-carboxylate oxidase genes[A]. The 2nd International Symposium on Biotechnology of Tropical and Subtropical Species[C]. Taipei: Institute of Botany Academia Sinica, 2001: 17.
[127] Chen M H, Wang P J, Maeda E. Somatic embryogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explants[J]. Plant Cell Reports, 1987, 6: 348-351.
[128] Chen G, Ye J, Huang M, et al. Cloning of the papaya ringspot Virus (PRSV) replicasegene of PRSV-resisitance through the introduction of the PRSV replicase gene[J]. Plant Cell Reports, 2001, 20(3): 272-277.
[129] Chenchik A, Zhu Y Y, Diatchenko L, et al. Generation and use of high quality cDNA from small amounts of total RNA by SMART PCR. In gene cloning and analysis by RT-PCR [D]. Biotechniques Books, 1998, 305-319.
[130] Cheng Y H, Yang J S, Yeh S D, et al. Efficient transformation of papaya by coat protein gene of papaya ringspot virus mediated by Agrobacterium following liquid-phase wounding of embryogenic tissues with caborundum[J]. Plant Cell Reports, 1996, 16: 127-132.
[131] Chibbar R N, Cella R, van Huystee R B. The heme moiety in peanut peroxidase[J]. Canadian journal of biochemistry and cell biology , 1984, 62(11): 1046-1050.
[132] Coux O, Tanaka K, Goldberg A L. Structure and function of the 20S and 26S proteasome[J]. Annu Rev Biochem, 1996, 65: 801-847.
[133] Crookers P R, Grierson D. Ultrastructure of tomato fruit ripening and the role of polygalacturonase isoenzymes in cell wall degradation[J]. Plant Physiology, 1983, 72: 1088- 1093.
[134] Domínguez-Puigjaner E, Llop-Tous I, Vendrell M, et al. A cDNA clone highly expressed in ripe banana fruit shows homology to pectate lyases [J]. Plant Physiology, 1997, 114(3): 1071-1076.
[135] Dong J G, Kim W T, Yip W K, et al. Cloning of a cDNA encoding 1-aminocyclopropane- 1-carboxylate synthase and expression of its mRNA in ripening apple fruit[J]. Planta, 1991,185(1): 38-45.
[136] Fischer R L, Bennett A B. Role of cell wall hydrolases in fruit ripening[J]. Annual Review of Plant Physiology and Plant Molecular Biology, 1991, 42: 675-703.
[137] Fitch M M M. High frequency somatic embryogenesis and plant regeneration from papaya hypocotyl callus[J]. Plant Cell, Tissue and Organ Culture, 1993, 32(2): 205-212.
[138] Fitch M M M, Manshardt R M. Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.)[J]. Plant Cell Reports, 1990, 9(6): 320-324.
[139] Fitch M M M, Manshardt R M, Gonsalves D, et al. Stable transformation of papaya via microprojectile bombardment[J]. Plant Cell Reports, 1990, 9(4): 189-194.
[140] Frohman M A, Dush M K, Martin G R. Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer[J]. Proc Natl Acad Sci USA, 1988, 85: 8998-9002.
[141] Fukuda T, Kido A, Kajino K, et al. Cloning of differentially expressed genes in highly and low metastatic rat osteosarcom as by a modified cDNA-AFLP method[J]. Biochemical and Biophysical Research Communications, 1999, 261(1): 35-40.
[142] Gasteiger E, Hoogland C, Gattiker A, et al. Protein Identification and Analysis Tools on the ExPASy Server. (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press[M]. 2005, 571-607.
[143] Geourjon C, Deléage G. SOPM: a self-optimised method for protein secondary structure prediction[J]. Protein Engineering, 1994, 7: 157-164.
[144] Gonsalves D. Control of papaya ringspot Virus in papaya: a case Study[J]. Annual Review Phytopathology, 1998, 36: 415-437.
[145] Grierson D, Tucker G A, Keen J, et al. Sequencing and identification of a cDNA clone for tomato polygalacturonase [J]. Nucleic Acids Research, 1986, 14 (21): 8595-8603.
[146] Habu Y, Fukada-Tanaka S, Hisatomi Y, et al. Amplified restriction fragment length polymorphism-based mRNA fingerprinting using a single restriction enzyme thatrecognizes a4-bp sequence[J]. Biochemical and Biophysical Research Communications, 1997, 234(2): 516-521.
[147] Hall L N, Bird C R, Picton S, et al. Molecular characterisation of cDNA clones representing pectin esterase isozymes from tomato[J]. Plant Mol Biol, 1994, 25: 313-318.
[148] Hall L N, Tucker G A, Smith C J S, et al. Antisense inhibition of pectin esterase gene expression in transgenic tomatoes[J]. Plant Journal, 1993, 3: 121-129.
[149] Hamilton A J, Bouzayen M, Grierson D. Identification of a tomato gene for the ethylene-forming enzyme by expression in Yeast[J]. Proc Natl Acad Sci, 1991, 88(16): 7434-7437.
[150] Hamilton A J, Lycett G W, Grierson D. Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants[J]. Nature, 1990, 346: 284-287.
[151] Hashimoto K, Sato N. Characterization of the mitochondrial NAD7 gene in Physcomitrella patens: similarity with angiosperm NAD7 genes[J]. Plant Science, 2001, 160: 807-815.
[152] Hidalgo M S, Tecson-Mendoza E M, Laurena A C, et al. Hybrid‘Sinta’papaya exhibits unique ACC synthase 1 cDNA isoforms[J]. Journal of Biochemistry and Molecular Biology, 2005, 38(3): 320-327.
[153] Hobson G E. The firmness of tomato fruit in relation to polyglacturonase activity[J]. J Hort Sci, 1965, 40: 66-72.
[154] Huber D J. The role of cell wall hydrolases in fruit softening[J]. Horticultural Reviews, 1983, 5: 169 -219.
[155] Ikoma Y, Yano M, Ogawa K. Cloning and expression of genes encoding ACC synthase in Kiwifruit[J]. Acta Horticulture, 1995, 398: 179-183.
[156] Inuzuka M, Hayakawa M, Ingi T. Serinc, an activity-regulated protein family, incorporates serine into membrane lipid synthesis[J]. J Biol Chem, 2005, 280(42): 35776-35783.
[157] Jiménez-Brtmudez S, Redondo-Nevado J, Mu?oz -Blanco J, et al. Manipulation of strawberry fruit softening by antisense expression of a pectatelyase gene[J]. Plant Physiology, 2002, 128: 751-759.
[158] Kende H. Ethylene biosynthesis[J]. Annu Rev Plant Physiol, Plant Mol Biol, 1993, 44: 283-307.
[159] Koch J L, Nevins D J. Tomato fruit cell wall. Part I. Use of purified tomato polygalacturonase and pectinmethylesterase to identify developmental changes in pectins [J]. Plant Physiology, 1989, 91: 816-822.
[160] Kotewicz M L, Sampson C M, Dalessio J M, et al. Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity[J]. Nucleic Acids Res, 1988, 16: 265-277.
[161] Lazan H, Ng S Y, Goh L Y, et al. Papayaβ-galactosidase/galactanase isoforms in differential cell wall hydrolysis and fruit softening during ripening[J]. Plant Physiology and Biochemistry, 2004, 42(11): 847-853.
[162] Lazan H, Selamat M K, Ail Z M.β-galactosidase, polygalacturonase and pectin esterase in differential softening and cell wall modification during papaya fruit ripening[J]. Physiologia Plantarum, 1995, 95(1): 106-112.
[163] Lee S A, Ross G S, Gardner R C. An apple (Malus domestica L. Borkh cv. Granny Smith) homolog of the ethylene receptor gene ETR1[J]. Plant Physiology, 1998, 117: 1125.
[164] Liang P, Pardee A B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction[J]. Science, 1992, 257(5072): 967-971.
[165] Lin C T, Fu K Y, Lin M T, et al. Cloning and characterization of a cDNA for 1-aminocyclopropane-1-carboxylate synthase from papaya fruit[J]. Plant Physiol, 1998, 116: 1193.
[166] Lin C T, Shaw J F, Lin M T. Cloning and characterization of a cDNA for 1-aminocyclopropane- 1-carboxylate oxidase from papaya fruit[J]. Journal of Agricultural and Food Chemistry, 1997, 45(2): 526-530.
[167] Lincoln J E, Campbell A D, Oetiker J, et al. LE-ACS4, a fruit ripening and wound-induced 1-aminocyclopropane-1-carboxylate synthase gene of tomato (Lycopersicon esculentum) [J]. J Biol Chem, 1993, 268(26): 19422-19430.
[168] litz R E, Conover R A. Development of systems for obtaining parasexual Carica hybrids[J]. Proc Fla State Hort Soc, 1979, 92: 180-182.
[169] litz R E, Conover R A. Somatic embryogenesis in cell cultures of Carica Stiputata[J]. Hort Science, 1980, 15: 733-735.
[170] Litz R E, Conover R A. In vitro somatic embryogenesis and plant regeneration from Carica papaya L. ovulaf callus[J]. Plant Sci Lett, 1982, 26: 153-158.
[171] Llop-Tous I, Domínguez-Puigjaner E, Palomer X, et al. Characterization of two divergent endo-β-1,4-glucanase cDNA clones highly expressed in the nonclimacteric strawberry fruit[J]. Plant Physiol, 1999, 119: 1415-1422.
[172] Lobarzewski J, Greppin H, Penel C, et al. Biochemical, molecular, and phyaiological aspects of plant peroxidases[M]. University M. CurieSklodowska, Lubin, Poland and University of Geneva, Switzerland, 1991: 3-13.
[173] Logemann J, Schell J, Willmitzer L. Improved method for the isolation of RNA from plant tissues[J]. Anal Biochemistry, 1987, 163(1): 16-20.
[174] Loschiavo F, Filippini F, Cozzani F, et al. Modulation of auxin-binding proteins in cell suspensions. I. Differential responses of carrot embryo cultures[J]. Plant Physiology, 1991, 97: 60-64.
[175] Lynd L R, Weimer P J, Paul J W, van Zyl W H, et al. Micribial cellulose utilization: fundamentals and biotechnology[J]. Microbiology and molecular biology reviews, 2002, 66(3): 506-577.
[176] Ma H, Moore P H, Liu Z Y, et al. High-density linkage mapping revealed suppression of recombination at the sex determination locus in papaya[J]. Genetics, 2004, 166: 419-436.
[177] Magdalita P M, Laurena A C, Yabut-Perez B M, et al. Progress in the development of transgenic papaya: transformation of Solo papaya using ACC synthase antisense construct[J]. Acta Horticulturae, 2002, 575: 171-176.
[178] Manning K. Isolation of nucleic acids from plants by differential solvent precipitation[J]. Anal Biochem, 1991, 195(1): 45-50.
[179] Manrique G D, Lajolo F M. FT-IR spectroscopy as a tool for measuring degree of methyl esterification in pectins isolated from ripening papaya fruit[J]. Postharvest biology and technology, 2002, 25: 99-107.
[180] Marchler-Bauer A, Anderson J B, Derbyshire M K, et al. CDD: a conserved domain database for interactive domain family analysis[J]. Nucleic Acids Res, 2007, 35: 237-240.
[181] Marín-Rodriguez M C. Investigation of the role of pectate lyase in banana fruit softening[D]. PhD dissertation, University of Greenwich, 2001.
[182] Mason M G, Botella J R. Identification and characterisation of two 1-aminocyclopropane -1-carboxylate (ACC) synthase cDNAs expressed during papaya (Carica papaya L.) fruit ripening[J]. Australian Journal of Plant Physiology, 1997, 24(2): 239-244.
[183] Matz M, Shagin D, Bogdanova E, et al. Amplification of cDNA ends based on template-switching effect and step-out PCR[J]. Nucleic Acid Res, 1999, 27(6): 1558-1560.
[184] Maunders, M J, Holdsworth, M J, Slater A, et al. Ethylene stimulates the accumulation of ripening-related mRNA in tomatoes[J]. Plant, Cell and Environment, 1987, 10(2): 177-184.
[185] Medina-Escobar N, Cárdenas J, Moyano E, et al. Cloning molecular characterisation and expression pattern of a strawberry ripening-specific cDNA with sequence homology to pectate lyase from higher plants[J]. Plant Molecular Biology, 1997, 34(6): 867-877.
[186] Miller W R, Mcdonald R E. Irradiation, stage of maturity at harvest, and storage temperature during ripening affect papaya fruit quality[J]. HortScience, 1999, 34(6): 1112-1115.
[187] Mita S, Kawamura S, Asai T. Regulation of the expression of a putative ethylene receptor, PeERS2, during the development of passion fruit (Passiflora edulis) [J]. Physiologia Plantarum, 2002, 114(2): 271-280.
[188] Money T, Reader S, Qu L J, et al. AFLP-based mRNA fingerprinting[J]. Nucleic Acids Research, 1996, 24: 2616-2617.
[189] Mondal M, Gupta S, Mekherhee B B. In vitro propagation of shoot buds of Carica papaya L. (caricaceae) Var. Honey Dew [J]. Plant Cell Reports, 1990, 8(10): 609-921.
[190] Moniz de S M, Drouin G. Phylogeny and substitution rates of angiosperm actin genes[J]. Mol Bio Evol, 1996, 13(9): 1198-1212.
[191] Murashige T, Skoog F. A rebised medium for rapid growth and bioassays with tobacco tissue cultures[J]. Physiol Plant, 1962, 15: 473-479.
[192] Muschietti J, Eyal Y, McCormick S. Pollen tube localization implies a role in pollen-pistil interactions for the tomato receptor-like protein kinases LePRK1 and LePRK2[J]. Plant Cell,1998, 10: 319-330.
[193] Neupane K R. Genetic engineering of papaya (Carica papaya L.) for modified ethylene biosynthesis[D]. Honolulu: University of Hawaii, PhD Dissertation, 1997.
[194] Neupane K R, Mukatira U T, Kato C, et al. Cloning and characterization of fruit-expressed ACC synthase and ACC oxidase from papaya (Carica papaya L.) [J]. Acta Horticulturae, 1998, 461: 329-337.
[195] Nunan K J, Davies C, Robinson S P, et al. Expression patterns of cell wall-modifying enzymes during grape berry development [J]. Planta, 2001, 214 (2): 257-264.
[196] Oeller P W, Lu M W, Taylor L P, et al. Reversible inhibition of tomato fruit senescence by antisense RNA[J]. Science, 1991, 254: 437-439.
[197] Olson D C, White J A, Edelman L, et al. Differential expression of two genes for 1-aminocyclopropane-1-carboxylate synthase in tomato fruits[J]. Proc Natl Acad Sci, 1991, 88(12): 5340-5344.
[198] Othman R, Choo T S, Ali Z M, et al. A full-length beta-galactosidase cDNA sequence from ripening papaya (Carica Papaya L. Cv. Eksotika)[J]. Plant Physiology, 1998, 118: 1102.
[199] Parasnis A S, Gupta V S, Tamhankar S A, et al. A highly reliable sex diagnostic PCR assay for mass screening of papaya seedlings[J]. Molecular Breeding, 2000, 6: 337-344.
[200] Parrott W A. Auxin-stimulated somatic embryogenesis from immature cotyledons of white clover[J]. Plant Cell Reports, 1991, 10(1): 17-21.
[201] Paull R E, Gross K, Qiu Y. Changes in papaya cell walls during fruit ripening[J]. Postharvest Biology and Technology, 1999, 16: 79-89.
[202] Picton S, Barton S L, Bouzayen M, et al. Altered fruit ripening and leaf senescence in tomatoes expressing an antisense ethylene-forming enzyme transgene[J]. Plant Journal, 1993, 3: 469-481.
[203] Pilatzke-Wunderlich I, Nessler C L. Expression and activityof cell-wall-degrading enzymes in the latex of opium poppy, Papaver somniferum L.[J]. Plant Molecular Biology, 2001, 45(5): 567-576.
[204] Price H J, Smith R H. Somatic embryogenesis in suspension cultures of G, klotzschianum Anderss[J]. Planta, 1979, 145: 305-307.
[205] Redgwell R J, Melton L D, Brasch D J. Cell wall dissolution in ripening kiwifruit (Actinidia deliciosa): solubilization of the pectic polymers[J]. Plant Physiology, 1992, 98(1): 71-81.
[206] Reuveni O, Shlesinger D R, Lavi U. In vitro clonal propagation of dioecious Carica papaya[J]. Plant Cell, Tissue and Organ Culture, 1990, 20 (1): 41-46.
[207] Ross G S, Knighton M L, Lay-Yee. An ethylene-related cDNA from ripening apples[J]. Plant Molecular Biology, 1992, 19(2): 231-238.
[208] Ross G S, Wegrzyn T, MacRae E A, et al. Apple beta-galactosidase activity against cell wall polysaccharides and characterization of a related cDNA clone[J]. Plant Physiology, 1994, 106 (2): 521-528.
[209] Sato T, Theologis A. Cloning the mRNA encoding 1-aminocyclopropane-1-carboxylate synthase, the key enzyme for ethylene biosynthesis in plants[J]. Proc Natl Acd Sci, 1989,86(17): 6621-6625.
[210] Sheehy R E, Kramer M, Hiatt W R. Reduction of polygalacturonase actibity in tomato fruit by antisense RNA[J]. Proc Natl Acad Sci, 1988, 85(23): 8805-8809.
[211] Slater A, Maunders M J, Edwards K, et al. Isolation and characterization of cDNA clones for tomato polygalacturonase and other ripening-related proteins[J]. Plant Molecular Biology, 1985, 5(3): 137-147.
[212] Smith D L, Abbott J A, Gross K C. Down-regulation of tomatoβ-galactosidase 4 results in decreased fruit softening[J]. Plant Physiology, 2000, 123: 173-183.
[213] Smith D L, Starrett D A, Gross K C. A gene coding for tomato fruitβ-galactosidase II is expressed during fruit ripening: Cloning, characterization, and expression pattern[J]. Plant Physiology, 1998, 117 (2): 417-423.
[214] Smith C J, Watson C F, Morris P C, et al. Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes[J]. Plant Molecular Biology, 1990, 14(3): 369-379.
[215] Smith C J, Watson C F, Ray J, et al. Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes[J]. Nature, 1988, 334: 724-726.
[216] Song W Y, Pi L Y, Wang G L, et al. Evolution of the rice Xa21 disease resistance gene family[J]. Plant Cell, 1997, 9(8): 1279-1287.
[217] Staiger C J, Schliwa M. Actin localization and function in higher plants[J]. Protoplasma, 1987,141(1): 1-12.
[218] Tateishi A, Inoue H, Shiba H, et al. Molecular cloning ofβ-galactosidase from Japanese pear (Pyrus pyrifolia) and its gene expression with fruit ripening[J]. Plant and Cell Physiology, 2001, 42 (5): 492-498.
[219] Tucker, G A, Grierson D. Synthesis of polygalacturonase during tomato fruit ripening[J]. Planta, 1982, 115(1): 64-67.
[220] Tucker, G A, Robertson N G, Grierson D. Changes in polygalacturonase isoenzymes during the ripening of normal and mutant tomato fruit[J]. European Journal Biochemistry, 1980, 112(1): 119-124.
[221] Vierstra R D. The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins[J]. Trends Plant Sci, 2003, 8(3): 135-142.
[222] Wang K L, Li H, Ecker J R. Ethylene biosynthesis and signaling networks[J]. Plant Cell, 2002, 14: S131-S151.
[223] Wheeler G L, Jones M A, Smirnoff N. The biosynthetic pathway of vitamin C in higher plants[J]. Nature, 1998, 393: 365-369.
[224] Wilkinson J Q, Lanahan M B, Clark D G, et al. A dominant mutat receptor from Arabidopsis confers ethylene insensitivity in heterologous plants[J]. Nature Biotechnology, 1997, 15: 444-447.
[225] Willats W G T, McCartney L, Mackei W, et al. Pectin: cell biology and prospects for functional analysis[J]. Plant Molecular Biology, 2001, 47(1-2): 9-27.
[226] Wool I G. Extraribosomal functions of ribosomal proteins [J]. Trends Biochem Sci, 1996, 21(5): 164-165.
[227] Woolley L C, James D J, Manning K. Purification and properties of an endo-beta-1,4-glucanase from strawberry and down-regulation of the corresponding gene, cel1[J]. Planta, 2001, 214: 11-21.
[228] Wu H T, Do Y Y, Huang P L. Nucleotide sequence of a cDNA encoding ethylene receptor from banana fruits[J]. Plant Physiology, 1999, 119(2): 805.
[229] Yang S F, Hoffman N E. Ethylene biosynthesis and its regulation in higher plants[J]. Annual Review of Plant Physiology, 1984, 35(1): 155-189.
[230] Yau C P, Wang L J, Yu M, et al. Differential expression of three genes encoding an ethylene receptor in rice during development, and in response to indole-3-acetic and silver ions[J]. Journal of Experimental Botany, 2004, 55: 547-556.
[231] Yie S T, Liaw S I. Plant regeneration from shoot tips and callus of papaya[J]. In Vitro Cellular and Developmental Biology, 1977, 13(9): 564-568.
[232] Yu E Y, Kim S E, Kim J H, et al. Sequence-specific DNA recognition by the Myb-like domain of plant telomeric protein RTBP1[J]. J Biol Chem, 2000, 275(31): 24208-24214.
[233] Zainon M A, Ng S Y, Othman R, et al. Isolation, characterization and significance of papayaβ-galactanases to cell wall modification and fruit softening during ripening[J]. Physiologia Plantarum, 1998, 104(1): 105-115.
[234] Zhuang J P, Su J, Li X P, et al. Cloning and expression analysis of beta-Galactosidase gene related to softening of banana (Musa sp.) Fruit[J]. Journal of Plant Physiology and Molecular Biology, 2006, 32(4): 411-419.