利用反义蜡质基因改良杂交稻食味品质的研究
|
摘要
本文在对我国杂交水稻的发展历程作了简要回顾的基础上，系统地分析了杂交稻目前的食味品质现状及淀粉、蛋白质、脂肪的含量，糊化温度，胶稠度，香味等因素对杂交稻的食味品质的影响。在影响稻米食味品质的诸多因素中，稻米直链淀粉含量是一项最重要的指标。由于控制直链淀粉含量的基因最主要的是蜡质基因，本文随后系统的对蜡质基因(Wx)及其等位基因位点进行分析，从顺式和反式两个方面对它的基因调控进行研究，除此之外，本文还对国内外利用反义蜡质基因(anti-Waxy)来改良稻米品质的情况进行了阐述，指出了系统研究蜡质基因、反义蜡质基因，来改良稻米蒸煮和食味品质的必要性和重要性。
本试验的主要工作是在根癌农杆菌介导下，应用携带反义蜡质基因的p13W4质粒分别对粳型三系杂交稻恢复系湘晴和三系不育系香粳进行遗传转化；同时应用携带反义Waxy基因的p13W4质粒和p13W8质粒对两系不育系261S尝试进行共转化研究。开展本项研究可望为人们今后利用基因工程技术改良杂交稻食味品质研究提供依据。
本文在分析了影响根癌农杆菌感染水稻和获得转基因水稻植株的各种因素后，建立了农杆菌介导的水稻高效转化体系。按此试验体系，湘晴抗性愈伤组织占总愈伤组织比例和能出苗的抗性愈伤组织比例分别可达到52.9％和16.4％；261S的共转化率达到56.5％。对三系杂交稻T_0代转基因植株进行PCR和Southern杂交检测(恢复系湘晴)，表明外源基因已经整合进湘晴水稻基因组中。湘晴水稻(亲本)直链淀粉含量检测结果是15.79％，大多数转基因后代的直链淀粉含量明显低于对照。对杂交水稻后代的种子的直链淀粉含量测定结果表明，杂交后代的直链淀粉含量最低的可降低到9.38％，这说明双亲都改造后，对杂交后代的直链淀粉含量降低是非常明显的。通过分析杂交稻子代的直链淀粉含量，我们将进一步研究反义蜡质基因对杂交稻后代的直链淀粉含量和食味品质的影响，为杂交稻的食味品质改良奠定基础。
After elementary study had been made about phylogeny of hybrid rice, we systemically analysed the actual status quo of the eating quality of hybrid rice and the influencing factors such as the content of amylose protein fat, pastification temperature, gel consistency, scent, etc. Because the content of amylose is the most important index among these factors and anti-Waxy gene is the primariest gene controlling the content of amylose, we systemically expatiated the Wx location and its equipotent genes. After researched on positive and negative regulation, we investigated all the developmental history to improve the eating quality of rice by using anti-Waxy gene in all over the world . In a word, systemically studies on Wx gene and anti-Waxy gene are necessary and important to improve the eating quality of rice.
It is the primary work of the experiment that we induced an anti-Waxy gene which is in the plasmid P13W4 by Agrobacterium-mediated into the high yield japonica restorer line Xiangqing (three-line hybrid rice )and the male sterile line Minyouxiangjing (three-line hybrid rice). Subsequently we induced two anti-Waxy genes which are respective in two plasmids P13W4 and P13W8 into the male sterile line 261S (two-line hybrid rice) by Agrobacterium-mediated. We hope to create some valuable method in improvement the quality of hybrid rice. Finally. In a word, we expect to produce some strains with high yield, high quality and inherited stability etc.
After compared many influencing factors in the trial, we established a high efficient method induced by agrobacterium.The percentage of hygromycin-resistant and the percentage of callus which can birth seedling reach 52.9% and 16.4%,the percentage of co-tranformation reached 56.5%. The PCR and Southern analysis (only in Xiangqing) showed the anti-Waxy gene had been transferred into genome of these hybrid rice and expressed. Compared the assay with comparison, we found that the amylase content obviously decreased in a majority of lines. The amylase content of offspring of hybrid rice even fall to 9.38%. These testified it was effective and feasible to reduce the amylase content by reconstructing parents. Finally , through the comparison of the cross between different strains, we further study the influence of anti-Waxy on the eating quality of hybrid rice. This is a foundation to improve the eating quality of hybrid rice.
本试验的主要工作是在根癌农杆菌介导下，应用携带反义蜡质基因的p13W4质粒分别对粳型三系杂交稻恢复系湘晴和三系不育系香粳进行遗传转化；同时应用携带反义Waxy基因的p13W4质粒和p13W8质粒对两系不育系261S尝试进行共转化研究。开展本项研究可望为人们今后利用基因工程技术改良杂交稻食味品质研究提供依据。
本文在分析了影响根癌农杆菌感染水稻和获得转基因水稻植株的各种因素后，建立了农杆菌介导的水稻高效转化体系。按此试验体系，湘晴抗性愈伤组织占总愈伤组织比例和能出苗的抗性愈伤组织比例分别可达到52.9％和16.4％；261S的共转化率达到56.5％。对三系杂交稻T_0代转基因植株进行PCR和Southern杂交检测(恢复系湘晴)，表明外源基因已经整合进湘晴水稻基因组中。湘晴水稻(亲本)直链淀粉含量检测结果是15.79％，大多数转基因后代的直链淀粉含量明显低于对照。对杂交水稻后代的种子的直链淀粉含量测定结果表明，杂交后代的直链淀粉含量最低的可降低到9.38％，这说明双亲都改造后，对杂交后代的直链淀粉含量降低是非常明显的。通过分析杂交稻子代的直链淀粉含量，我们将进一步研究反义蜡质基因对杂交稻后代的直链淀粉含量和食味品质的影响，为杂交稻的食味品质改良奠定基础。
After elementary study had been made about phylogeny of hybrid rice, we systemically analysed the actual status quo of the eating quality of hybrid rice and the influencing factors such as the content of amylose protein fat, pastification temperature, gel consistency, scent, etc. Because the content of amylose is the most important index among these factors and anti-Waxy gene is the primariest gene controlling the content of amylose, we systemically expatiated the Wx location and its equipotent genes. After researched on positive and negative regulation, we investigated all the developmental history to improve the eating quality of rice by using anti-Waxy gene in all over the world . In a word, systemically studies on Wx gene and anti-Waxy gene are necessary and important to improve the eating quality of rice.
It is the primary work of the experiment that we induced an anti-Waxy gene which is in the plasmid P13W4 by Agrobacterium-mediated into the high yield japonica restorer line Xiangqing (three-line hybrid rice )and the male sterile line Minyouxiangjing (three-line hybrid rice). Subsequently we induced two anti-Waxy genes which are respective in two plasmids P13W4 and P13W8 into the male sterile line 261S (two-line hybrid rice) by Agrobacterium-mediated. We hope to create some valuable method in improvement the quality of hybrid rice. Finally. In a word, we expect to produce some strains with high yield, high quality and inherited stability etc.
After compared many influencing factors in the trial, we established a high efficient method induced by agrobacterium.The percentage of hygromycin-resistant and the percentage of callus which can birth seedling reach 52.9% and 16.4%,the percentage of co-tranformation reached 56.5%. The PCR and Southern analysis (only in Xiangqing) showed the anti-Waxy gene had been transferred into genome of these hybrid rice and expressed. Compared the assay with comparison, we found that the amylase content obviously decreased in a majority of lines. The amylase content of offspring of hybrid rice even fall to 9.38%. These testified it was effective and feasible to reduce the amylase content by reconstructing parents. Finally , through the comparison of the cross between different strains, we further study the influence of anti-Waxy on the eating quality of hybrid rice. This is a foundation to improve the eating quality of hybrid rice.
引文
1． 徐庆国．日本关于稻米品质的研究Ⅱ．国外农学——水稻，1985，(3)：1-6
2． 周坤炉，白德朗，阳和华．杂交香稻香味的遗传与应用．湖南农业科学．1989(2)：10-12
3． 宋光泉、陈清泉．精米中氮磷钾钠的含量与品质特征的关系．湖南农业科学，1989(3)：21-23
4． 武小金．稻米蒸煮品质性状的遗传研究．湖南农学院学报．1989，15(4)：6—10
5． 向远鸿，唐启源，黄燕湘．稻米品质性状相关性研究：籼型粘稻食味与其它米质性状的关系．湖南农学院学报，1990，16(4)：325-330
6． 刘宜柏．水稻优质资源的筛选及品质研究的综合报告．江西农业大学学报，1991，13(4)：310-315
7． 陈清泉，宋光泉，欧阳静仁．籼型杂交稻与常规稻米质特性差异的研究．中国农业科学，1991，24(2)：43—50
8． 华健，王宗阳，张景六等．核蛋白因子与水稻蜡质基因5’端上游顺序的结合．科学通报，1992，18：1698
9． 易小平，陈芳远．籼型杂交水稻稻米蒸煮品质，碾米品质及营养品质的细胞质遗传效应．中国水稻科学，1992，6(4)．-187-189
10． 萨姆布鲁克．费里奇，等编．分子克隆实验指南(第二版)．1992，科学出版社
11． 何予卿，吕志仁．籼稻米直链淀粉含量的遗传及其基因剂量效应．华中农业大学学报，1993，12(5)：414—420
12． 孙宗修 斯华敏，程式华，等．麦芽糖提高水稻花药培养效率的研究．中国水稻科学，1993，7(4)：227-231
13． 任光俊，李青茂，彭兴富，等．水稻香味的遗传．四川农业大学学报，1994，12(3)：392—395
14． 刘庆荣，等．浙江省杂交水稻米质现状和改良途径．杂交水稻，1995，(2)：28—32
15． 李欣，汤述翥，陈宗祥，等．粳稻米糊化温度的遗传研究．江苏农学院学报，1995，16(1)：15-20
16． 徐庆国．稻米品质的品种种性差异研究．湖南农业大学学报，1995，21(4)：337-341
17． 徐辰武，莫惠栋，张爱红，等．籼—粳杂种稻米品质性状的遗传控制．遗传学报，1995，22(3)：192—198
18． 石春海，朱军．稻米营养品质种子效应和母体效应的遗传分析．遗传学报，1995，22(5)：372—379
19． 石春海，杨肖娥．种子，细胞质和母体遗传效应对籼型杂交稻稻米营养品质杂交优势的影响．杂交水稻．1996(1)．23-27
20． 陈丽，王宗阳，张景六，等．水稻蜡质基因5’端上游顺序中的核蛋白结合区．植物生理学报，1996，22(4)：349—356
21． 徐辰武，莫惠栋．籼稻米糊化温度的质量—数量遗传分析．作物学报，1996，22(4)．384-391
22． 姚彩萍，王宗阳．水稻蜡质基因5’上游区缺失对基因表达的影响．植物生理学报，1996，22(4)：431-436
23． 王长发，高如嵩．籼型杂交水稻稻米糊化温度的遗传分析．西北农业大学学报，1996，24(1)：28-32
24． 蔡秀玲，王宗阳．郑霏琴，等．水稻蜡质基因5’非翻译区一个与调控有关的内含子．植物生理学报，1997，23(3)：257—261
25． 徐正进，等．水稻品质性状的品种间差异及其产量关系的研究．沈阳农业大学学报，1997，10(3)：29-34
26． 梁满中，Zaman F U, Dikshit H K．杂交水稻直链淀粉含量遗传分析．生命科学研究，1998，2(3)：224—228
27． 刘巧泉，张景六．根癌农杆菌介导的水稻高效转化系统的建立．植物生理学报，1998，24(3)：259-271
28． 舒庆尧，Mccl.An．稻米淀粉RVA谱特征与食用品质的关系．中国农业科学，1998，31(3)：25-29
29． 何昆明，李晓方，林红，等．水稻产量和品质若干构成因素的遗传分析．广东农业科学，1998(增刊)：26—28
30． 仲维功，陈志德，朱述钧．优质米水稻品种的选育与特点．江苏农业科学，1999(1)：14-16
31． 黄慧君，黄道强．山梨醇对水稻体细胞培养的特殊作用．中国水稻科学，1999，13(4)：248-250
32． 廖伏明 周坤炉．籼型三系杂交水稻米质现状研究．杂交水稻，1999，14(6)：35-38
33． 项友斌，梁竹青，高明尉，等．农杆菌介导的苏云金杆菌基因CryIACb和CryIA(C)在水稻中的遗传转化及蛋白表达．生物工程学报，1999，15(4)：494-500
34． 章冰，卫志明．根癌农杆菌介导的水稻转化及转基因Rl代植株表型特征．植物生理学报，1999，25(4)：313-320
35． 刘志学，马向前，何艺园．农杆菌介导遗传转化中辅助处理方法的改良．复旦学报(自然科学版)，1999，38(5)：601-604
36． 刘志学，张旭，徐亚南，等．用LBA4404／pCAMBIA系列转化水稻的最佳条件．复旦学报(自然科学版)，1999，38(4)：439-443
37． 王小兰，黄占景，沈银柱．反义蜡质基因质粒pWXAB的构建．河北师范大学学报(自然科学版)，2000，24(1)：105-105
38． 蔡秀玲，王宗阳，邢彦彦，等．水稻蜡质基因第一内含子碱基突变引起其RNA二级结构的可能变化．植物生理学报，2000，26(1)：59-63
39． 唐威华，王宗阳，张景六，等．水稻蜡质基因第一内含子中g片段上核蛋白因子结合位点的确定．植物生理学报，2000，26(4)：323-330
40． 黄祖六，谭学林，等．稻米直链淀粉含量基因座位的标记定位．作物学报，2000，26(6)：777-782
41． 张爱民，肖兴国，聂秀玲．中国的植物转基因雄性不育研究．中国科学基金，2000，3：32-135
42． 罗闰良，吴京华．杂交稻及其发展．科学，2000，52(6)：51-53
43． 包劲松，朱立煌．水稻Wx基因(CT)_n微卫星标记与稻米淀粉品质的关系研究．农业生物技术学报，2000，8(3)：241-244
44． 卢庆善，孙毅，华泽田．农作物杂种优势．北京：中国农业科技出版社，2001，10：240-268
45． 戚华雄，陈志军，等．稻米直链淀粉含量的分子检测技术．湖北农业科学．2001，5：3-5．
46． 陈秀花，刘巧泉，王宗阳．反义Wx基因导入我国籼型杂交稻重点亲本．科学通报，2002，(9)：684-688
47． 林鸿生，李娜，等．农杆菌介导glgC-TM基因转化水稻研究．中国水稻科学，2002，16(2)：129-133
48． 王宗阳，顾铭洪，等．根癌农杆菌介导的水稻转化系统的优化及转反义Wx基因植株的获得．中国水稻科学，2002，16(4)：304-310
49． 殷丽青，沈革志，等．用根癌农杆菌介导获得籼稻转基因植株．上海农业学报，2002．18(1)：16-19
50． 李秀兰，吴成，邓晓建，等．生物技术改良稻米淀粉品质的进展．中国农学通报，2002，18，(3)：61-64，119
51． 庞华莒．不同类型水稻品质性状分析．中国农学通报，2002，18(3)：37-40
52． 陈跃进．水稻直链淀粉含量的分子遗传研究．湖南农业大学学报，2002，28(3)：195-198
53． 张小明，王仪春，石春海．等．稻米蒸煮营养品质性状的遗传研究进展．植物遗传资源科学，2002，3(2)：51—55
54． 范士靖．上海师范大学硕士学位论文，2002
55． 黄少军，梁庆平．早籼杂交稻稻米品质性状相关分析．西北农业科学，2003，1：15-17
56． 刘峰，许明，陈福禄，等．无选择标记的高光效基因植物表达载体的构建．福建农林大学学报(自然科学版)，2003，32(2)：270-272
57． 郭咏梅，卢义宣，刘晓利，等．杂交籼稻稻米主要品质性状的遗传改良．西南农么学报，2003，16(2)：17-21
58. Aldemita RR, Hodges TK. Agrobacterium tumefaciens-mediated transfomation of jacanica and indica rice varieties. Planta, 1996,199:612—617
59. Ali S S, Jafri S J H, Khan M G, et al. Inheritance studies for aroma in two aromatic varieties of Pakistan.IRRI, 1993,18(2):6
60. Amano E. Genetic and biochemical characterization of Wx mutants in cereals Environ Health Perspect. 1981, Jan;37:35-41
61. Ayres N M, McClung AM, Larkin P D, et al. Microsatellites and a single nucleotide polymorphism differentiate apparent amylose classes in an extended pedigree of US rice germplasm. Theor Appl Genet, 1997,94:773-781
62. Bao J S, Zheng X W,Xia Y W, et al. QTL mapping for the paste viscosity characteristics in rice (Oryza sativas L.) Theor Appl Genet, 2000,98:502-508
63. Berner D K, Hoff B J. Inheritance of scent in American long grain rice. Crop Sci, 1986,26:876-878
64. Bligh H F,Trill R, Jones C A.A microsatellite sequence closely linked to the Wx gene of Oryza sativa. Euphytica, 1995,86:83-85
65. Bligh HH, Larkin PD, Riach PS, et al. Use of alternate splice sites in granule-bound starch synthase MRNA from low-amylose rice varieties. Plant Mol Biol. 1998,38:407-415
66. Cai XL,Wang ZY, Xing YY, et al. Aberrant splicing of intron I leads to the heterogenesis 5 UTR and decreased expression of Wx gene in rice cultivars of intermediate amylase content. Plant J. 1998,14:459-465
67. Chang W L, Li W Y. Inheritence of Amylose Content and Gel Consistency in Rice. Bot. Bull of Academica Sinica, 1981,22(1):35-47
68. Clark JR. Robertson M, Ainsworth CC. Nucleotide sequence of a wheat(Triticum aestivum L.)cDNA clone encoding the Wx protein. Plant Mol Biol,1991, 16:1099-1101
69. De Neve M, de Buck S, Jacohs A, et al. T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of T-DNAs. Plant J, 1997, 11: 1286-1292
70. Dedolph, C. New directions in rice crop and seed improvement World Agriculture
1993,15-18
71. Dillen W, De Chercq J, Kapila J. et al. The effect of temperature on Agrobacterium-mediated gene transfer to plants, Plant J, 1997,2:1459-1463
72. Dong Yanjun, Zhang Hongde, Shi Shouyun. Genetic studies of aroma in the elite cytoplasmic male sterile aromatic japonica line Shanghai A. IRRI, 1992,17(2):5
73. Feldmann, KA.T-DNA insertion mutagenesis in Arabidopsis. mutational spectrum. Plant J. 1991, 1:71 82
74. Frances H, Bligh J, Larkin PD, et al. Use of alternate splice sites in granule-bound starch synthase mRNA from low-amylose rice varieties Plant Mol Biol. 1998,Oct;38(3):407-15
75. Goto F, Yoshihara T, Shigemoto N, et al. Iron fortification of rice seed by the soybean ferritin gene. Nat biotechnol. 1999,17(3):282-286
76. Hiei Y, Ohta S, Koomari T, et al. Efficient transformation of rice (Oryza sativa L.)mediated by Agrobacterrum and sequence analysis of the boundaries of the T-DNA. Plant J, 1994,6:271-282
77. Hirano H, Sano Y. Molecular characterization of the Waxy locus of rice(Oryza sativa).Plant Cell Physiol. 1991,32:989
78. Hirano HY, Eiguchi M, Sano Y.A single base change altered the regulation of the Wx gene at the posttranscriptional level during the domestrication of rice. Mol Biol Evol, 1998,15:978-987
79. Hirano HY, Sano Y. Comparison of Wx gene regulation in the endosperm and pollen in Oryza sativa L. Genes Genet Syst. 2000, Oct;75(5):245-9
80. Hsieh S C, et al. Evaluation and Genetic Studies on Grain Quality Characters in Rice. Symp. Plant Breed, 1982:99-112
81. Ikeno S. über die Bestaubung und die Bastardierung von Reis. Z Pflanzenzü cht, 1914,2:495-503
82. Isshiki M,Morino K, Nakajima M,et al.A naturally occurring functional allele of the rice Wx locus has a GT to TT mutant at the 5 splice site of the first intron. Plant J, 1998,15:133-321
83. Isshiki M,Nakajima M,Satoh H, et al. Dull rice mutants with tissue-specific effects on the splicing of the Wx pre-Mrna[J]Plant J, 2000,23 (4):451-460
84. ItohK, Ozaki H, Okada K, et al. Introduction of Wx transgene into rice wx mutants leads to both high-and low-amylose rice Plant Cell Physiol. 2003, May;44(5):473-480
85. Jefferson RA. Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep, 1987, 5:387-405
86. Juliano BO. A simplified assay for milled rice amylase. Cereal, 1971, (16):334-340,360
87. Kavi Kishor PB, Reddy GM. Regeneration of rice plants from long-term root and embryo-derived callus culture. Current Sci, 1986,55(14):664-665
88. Khush G. S, Singh R.J,. Sur S. C, et al. Primary trisomics of rice origin, Morphology, cytology and use in linkage mapping. Genetics, 1984,107:141-167
89. Khush G.S. et al. Grain quality consideration in hybrid rice. International
symposium on hybrid Rice. 6-10, October, 1986, Changsha, Hunan, China
90. Khush G.S. Proceedings of the International symposium on hybrid rice. Hybrid Rice, IRRI, Manila. 1988,39-54,201-215
91. K1sgen RJ, Gierl A, Schwarz-Sommer Z, et al. Molecular analysis of the Wx locus of Zea mays. Mol GenGenet, 1986,203:237-244
92. Komari T, Hiei Y, Saito Y, et al. Vectors carrying two separate T-DNAs for cn transformation of higher plants mediated by Agrobacteruim tumefaciens and segregation of transformants free from selection markers. Plant J, 1996, 10(1):165-174
93. Kumar et al. Genetic analysis of different amylose levels in rice. Crop Sci, 1987, (27):1167-1172
94. Kumar I, Khush G S, Juliano BO. Benetic analysis of Wx locus in rice (Oryza sativa L).Theor Appl Genet, 1987,73:481-488
95. Kumria R. Plant regeneration form transformed embryogenie calus of an elite indica rice via Agrobacterium. Plant Cell, Tissus, and Organ Culture, 2001,67:63-67
96. Labra M, Savini C, Bracale M,et al. Genomic changes in transgenie rice(Oryza sativa L.)plants produced by infecting calli with Agrobacterium tumefaciens. Plant Cell Reports. 2001,20:325-330
97. Larkin,P. D., and Park, W.D. Transcript accumulation and utilization of alternate and non-consensus splice sites in rice granule-bound starch synthase are temperature-sensitive and controlled by a single-nucleotide polymorphism. Plant Mol. Biol. 1999,40:719-727
98. Liu Q, Wang Z, Cheng X, et al. Stable inheritance of the antisence Wx gene in transgenic rice with reduced amylose level and improved quality. Transginic Res. 2003Feb;12(1):71-82
99. Martin C and Smith, AM. Starch biosynthesis. Plant Cell. 1995,7:971-985
100. McCormac AC, Fowler MR, Chen DF, et al. Efficient co-transformation of Nicotiana tabacum by two independent T-DNAs, the effect of T-DNA size and implications for genetic separation. Trans Res, 2001, 10:143-155
101. Mikami I, Effects of the two common Wx alleles on different genetic backgrounds in rice. Plant Breed, 2000, 119(6): 505—508
102. Mikami I. Effects of the two common Wx alleles on different genetic backgrounds in rice. Plant Breed, 2000,119(6):505—508
103. Muraue M. The Advance of Rice Taste Evaluation. Tokyo:Japanese Farming News Press, 1997
104. Okagaki RJ,Wessler SR. Comparison of non-mutant and mutant Wx genes in rice and maize. Genetics, 1988,120:1137-1143.
105. Pearson L,Meagher RB. Diverse soybean actin transcripts contain a large intron in the 5 untranslated leader structural similarity to vertebrate muscle actin genes. Plant Mol Biol, 1990,14:513.
106. Pooni H S, et al. A comprehensive model for disomically inherited metrical traits expressed in triploid tissues. Heredity, 1992,62:166-174
107. Puri,R.P.,Siddg, E.A. Inheritance of gelatinization temperature inrice. Ind.J.
Genet. & P1 .Breed, 1980,40(2):450-455
108. Riker, AJ, Lyneis,MM.Locke, S.B. Comparative physiology of crown gall, attenuated crown gall, radiobacter, and hair root bacteria. Phytopathology, 1941,31,964-977
109. Rohde W, Becker D, Salamini F.Structural analysis of the Wx locus from Hordeum vulgare. Nucleic Acids Res 1988, 16:7185—7186
110. Sano Y. Differential regulation of Wx gene expression in rice endosperm. Theor Appl Genet, 1984,68:467-473
111. Sano, Y, Katsumata, M. and Okuno, K. Genetic studies of speciation in cultivated rice. 5. Inter- and intraspecific differentiation in the Wx gene expression of rice. Euphytica, 1985,35:1-9
112. Shimada H, Tada Y, Kawasaki T, et al. Antisense regulation of the rice Wx gene expression using a PCR-amplified fragment of the rice genome reduces the amylase content in grain starch. Theor Appl Genet, 1993,86:665-672
113. Sood B G, Siddiq E A.A rapid technique for scent determination in rice. Crop Sci, 1978,38:268-271
114. Stansel, J.W.The influence of heredity and environment on endosperm characteristics of rice(Oryza sativa L.).Dissertation Abstr. 1996,27:4813
115. Tan Y F. The three important traits for cooking and eating quality of rice grains are controlled by a singlelocus in an elite rice hybrid, shanyou 63. Theoretical and Applied Genetics, 1999, 99(3/4): 642—648
116. Terada R, Nakajima M, Isshiki M, et al. Antisense Wx genes with highly active promoters effectively suppress Wx gene expression in transgenic rice. Plant Cell Physiol. 2000, Jul;41(7):881-8
117. Tomar J B. Genetic Analysis of Amylose Content in Rice. Genetic Agrar, 1987, 41(3): 235—242
118. Vander Krol AR,Mol JN, Stuitje AR. Antisense genes in plants:an overview. Gene,1988 Dec 10,72(1-2):45-50
119. Vasil. V. and Z K, Vasil. Cell culture and somatic cell genetics of plant,In"Induction and Maintenance of Embryogenic callus cultures of cramineae"1984, pp36-42, Academic Press, Orlando, Florida
120. Wang ZY,Zheng FQ, Shen GZ, et al. The amylose content in rice endosperm is related to the post-transcrisptional regulation of the Wx gene. Plant J. 1995 Apr; 7(4):613-22
121. Wang, Z.Y, Zheng, F.Q, Wang, X.Q, et al. Characterization of two transposon like elements in rice Wx gene. Sci. China Series B. 1994,37:437-447
122. Zhou PH, Tan YF, He YQ, et al. Simultaneous improvement for four quality traits of Zhenshan97, an elite parent of hybrid rice,by molecular marker-assisted selection Theor Appl Genet. 2003, Jan;106(2):326-31
123. Zhu Y, Cai XL, Wang ZY, et al. An interaction between a MYC protein and EREBP protein is involved in transcriptional regulation of the rice Wx gene. Biol Chem. 2003,278(48):47803-11
2． 周坤炉，白德朗，阳和华．杂交香稻香味的遗传与应用．湖南农业科学．1989(2)：10-12
3． 宋光泉、陈清泉．精米中氮磷钾钠的含量与品质特征的关系．湖南农业科学，1989(3)：21-23
4． 武小金．稻米蒸煮品质性状的遗传研究．湖南农学院学报．1989，15(4)：6—10
5． 向远鸿，唐启源，黄燕湘．稻米品质性状相关性研究：籼型粘稻食味与其它米质性状的关系．湖南农学院学报，1990，16(4)：325-330
6． 刘宜柏．水稻优质资源的筛选及品质研究的综合报告．江西农业大学学报，1991，13(4)：310-315
7． 陈清泉，宋光泉，欧阳静仁．籼型杂交稻与常规稻米质特性差异的研究．中国农业科学，1991，24(2)：43—50
8． 华健，王宗阳，张景六等．核蛋白因子与水稻蜡质基因5’端上游顺序的结合．科学通报，1992，18：1698
9． 易小平，陈芳远．籼型杂交水稻稻米蒸煮品质，碾米品质及营养品质的细胞质遗传效应．中国水稻科学，1992，6(4)．-187-189
10． 萨姆布鲁克．费里奇，等编．分子克隆实验指南(第二版)．1992，科学出版社
11． 何予卿，吕志仁．籼稻米直链淀粉含量的遗传及其基因剂量效应．华中农业大学学报，1993，12(5)：414—420
12． 孙宗修 斯华敏，程式华，等．麦芽糖提高水稻花药培养效率的研究．中国水稻科学，1993，7(4)：227-231
13． 任光俊，李青茂，彭兴富，等．水稻香味的遗传．四川农业大学学报，1994，12(3)：392—395
14． 刘庆荣，等．浙江省杂交水稻米质现状和改良途径．杂交水稻，1995，(2)：28—32
15． 李欣，汤述翥，陈宗祥，等．粳稻米糊化温度的遗传研究．江苏农学院学报，1995，16(1)：15-20
16． 徐庆国．稻米品质的品种种性差异研究．湖南农业大学学报，1995，21(4)：337-341
17． 徐辰武，莫惠栋，张爱红，等．籼—粳杂种稻米品质性状的遗传控制．遗传学报，1995，22(3)：192—198
18． 石春海，朱军．稻米营养品质种子效应和母体效应的遗传分析．遗传学报，1995，22(5)：372—379
19． 石春海，杨肖娥．种子，细胞质和母体遗传效应对籼型杂交稻稻米营养品质杂交优势的影响．杂交水稻．1996(1)．23-27
20． 陈丽，王宗阳，张景六，等．水稻蜡质基因5’端上游顺序中的核蛋白结合区．植物生理学报，1996，22(4)：349—356
21． 徐辰武，莫惠栋．籼稻米糊化温度的质量—数量遗传分析．作物学报，1996，22(4)．384-391
22． 姚彩萍，王宗阳．水稻蜡质基因5’上游区缺失对基因表达的影响．植物生理学报，1996，22(4)：431-436
23． 王长发，高如嵩．籼型杂交水稻稻米糊化温度的遗传分析．西北农业大学学报，1996，24(1)：28-32
24． 蔡秀玲，王宗阳．郑霏琴，等．水稻蜡质基因5’非翻译区一个与调控有关的内含子．植物生理学报，1997，23(3)：257—261
25． 徐正进，等．水稻品质性状的品种间差异及其产量关系的研究．沈阳农业大学学报，1997，10(3)：29-34
26． 梁满中，Zaman F U, Dikshit H K．杂交水稻直链淀粉含量遗传分析．生命科学研究，1998，2(3)：224—228
27． 刘巧泉，张景六．根癌农杆菌介导的水稻高效转化系统的建立．植物生理学报，1998，24(3)：259-271
28． 舒庆尧，Mccl.An．稻米淀粉RVA谱特征与食用品质的关系．中国农业科学，1998，31(3)：25-29
29． 何昆明，李晓方，林红，等．水稻产量和品质若干构成因素的遗传分析．广东农业科学，1998(增刊)：26—28
30． 仲维功，陈志德，朱述钧．优质米水稻品种的选育与特点．江苏农业科学，1999(1)：14-16
31． 黄慧君，黄道强．山梨醇对水稻体细胞培养的特殊作用．中国水稻科学，1999，13(4)：248-250
32． 廖伏明 周坤炉．籼型三系杂交水稻米质现状研究．杂交水稻，1999，14(6)：35-38
33． 项友斌，梁竹青，高明尉，等．农杆菌介导的苏云金杆菌基因CryIACb和CryIA(C)在水稻中的遗传转化及蛋白表达．生物工程学报，1999，15(4)：494-500
34． 章冰，卫志明．根癌农杆菌介导的水稻转化及转基因Rl代植株表型特征．植物生理学报，1999，25(4)：313-320
35． 刘志学，马向前，何艺园．农杆菌介导遗传转化中辅助处理方法的改良．复旦学报(自然科学版)，1999，38(5)：601-604
36． 刘志学，张旭，徐亚南，等．用LBA4404／pCAMBIA系列转化水稻的最佳条件．复旦学报(自然科学版)，1999，38(4)：439-443
37． 王小兰，黄占景，沈银柱．反义蜡质基因质粒pWXAB的构建．河北师范大学学报(自然科学版)，2000，24(1)：105-105
38． 蔡秀玲，王宗阳，邢彦彦，等．水稻蜡质基因第一内含子碱基突变引起其RNA二级结构的可能变化．植物生理学报，2000，26(1)：59-63
39． 唐威华，王宗阳，张景六，等．水稻蜡质基因第一内含子中g片段上核蛋白因子结合位点的确定．植物生理学报，2000，26(4)：323-330
40． 黄祖六，谭学林，等．稻米直链淀粉含量基因座位的标记定位．作物学报，2000，26(6)：777-782
41． 张爱民，肖兴国，聂秀玲．中国的植物转基因雄性不育研究．中国科学基金，2000，3：32-135
42． 罗闰良，吴京华．杂交稻及其发展．科学，2000，52(6)：51-53
43． 包劲松，朱立煌．水稻Wx基因(CT)_n微卫星标记与稻米淀粉品质的关系研究．农业生物技术学报，2000，8(3)：241-244
44． 卢庆善，孙毅，华泽田．农作物杂种优势．北京：中国农业科技出版社，2001，10：240-268
45． 戚华雄，陈志军，等．稻米直链淀粉含量的分子检测技术．湖北农业科学．2001，5：3-5．
46． 陈秀花，刘巧泉，王宗阳．反义Wx基因导入我国籼型杂交稻重点亲本．科学通报，2002，(9)：684-688
47． 林鸿生，李娜，等．农杆菌介导glgC-TM基因转化水稻研究．中国水稻科学，2002，16(2)：129-133
48． 王宗阳，顾铭洪，等．根癌农杆菌介导的水稻转化系统的优化及转反义Wx基因植株的获得．中国水稻科学，2002，16(4)：304-310
49． 殷丽青，沈革志，等．用根癌农杆菌介导获得籼稻转基因植株．上海农业学报，2002．18(1)：16-19
50． 李秀兰，吴成，邓晓建，等．生物技术改良稻米淀粉品质的进展．中国农学通报，2002，18，(3)：61-64，119
51． 庞华莒．不同类型水稻品质性状分析．中国农学通报，2002，18(3)：37-40
52． 陈跃进．水稻直链淀粉含量的分子遗传研究．湖南农业大学学报，2002，28(3)：195-198
53． 张小明，王仪春，石春海．等．稻米蒸煮营养品质性状的遗传研究进展．植物遗传资源科学，2002，3(2)：51—55
54． 范士靖．上海师范大学硕士学位论文，2002
55． 黄少军，梁庆平．早籼杂交稻稻米品质性状相关分析．西北农业科学，2003，1：15-17
56． 刘峰，许明，陈福禄，等．无选择标记的高光效基因植物表达载体的构建．福建农林大学学报(自然科学版)，2003，32(2)：270-272
57． 郭咏梅，卢义宣，刘晓利，等．杂交籼稻稻米主要品质性状的遗传改良．西南农么学报，2003，16(2)：17-21
58. Aldemita RR, Hodges TK. Agrobacterium tumefaciens-mediated transfomation of jacanica and indica rice varieties. Planta, 1996,199:612—617
59. Ali S S, Jafri S J H, Khan M G, et al. Inheritance studies for aroma in two aromatic varieties of Pakistan.IRRI, 1993,18(2):6
60. Amano E. Genetic and biochemical characterization of Wx mutants in cereals Environ Health Perspect. 1981, Jan;37:35-41
61. Ayres N M, McClung AM, Larkin P D, et al. Microsatellites and a single nucleotide polymorphism differentiate apparent amylose classes in an extended pedigree of US rice germplasm. Theor Appl Genet, 1997,94:773-781
62. Bao J S, Zheng X W,Xia Y W, et al. QTL mapping for the paste viscosity characteristics in rice (Oryza sativas L.) Theor Appl Genet, 2000,98:502-508
63. Berner D K, Hoff B J. Inheritance of scent in American long grain rice. Crop Sci, 1986,26:876-878
64. Bligh H F,Trill R, Jones C A.A microsatellite sequence closely linked to the Wx gene of Oryza sativa. Euphytica, 1995,86:83-85
65. Bligh HH, Larkin PD, Riach PS, et al. Use of alternate splice sites in granule-bound starch synthase MRNA from low-amylose rice varieties. Plant Mol Biol. 1998,38:407-415
66. Cai XL,Wang ZY, Xing YY, et al. Aberrant splicing of intron I leads to the heterogenesis 5 UTR and decreased expression of Wx gene in rice cultivars of intermediate amylase content. Plant J. 1998,14:459-465
67. Chang W L, Li W Y. Inheritence of Amylose Content and Gel Consistency in Rice. Bot. Bull of Academica Sinica, 1981,22(1):35-47
68. Clark JR. Robertson M, Ainsworth CC. Nucleotide sequence of a wheat(Triticum aestivum L.)cDNA clone encoding the Wx protein. Plant Mol Biol,1991, 16:1099-1101
69. De Neve M, de Buck S, Jacohs A, et al. T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of T-DNAs. Plant J, 1997, 11: 1286-1292
70. Dedolph, C. New directions in rice crop and seed improvement World Agriculture
1993,15-18
71. Dillen W, De Chercq J, Kapila J. et al. The effect of temperature on Agrobacterium-mediated gene transfer to plants, Plant J, 1997,2:1459-1463
72. Dong Yanjun, Zhang Hongde, Shi Shouyun. Genetic studies of aroma in the elite cytoplasmic male sterile aromatic japonica line Shanghai A. IRRI, 1992,17(2):5
73. Feldmann, KA.T-DNA insertion mutagenesis in Arabidopsis. mutational spectrum. Plant J. 1991, 1:71 82
74. Frances H, Bligh J, Larkin PD, et al. Use of alternate splice sites in granule-bound starch synthase mRNA from low-amylose rice varieties Plant Mol Biol. 1998,Oct;38(3):407-15
75. Goto F, Yoshihara T, Shigemoto N, et al. Iron fortification of rice seed by the soybean ferritin gene. Nat biotechnol. 1999,17(3):282-286
76. Hiei Y, Ohta S, Koomari T, et al. Efficient transformation of rice (Oryza sativa L.)mediated by Agrobacterrum and sequence analysis of the boundaries of the T-DNA. Plant J, 1994,6:271-282
77. Hirano H, Sano Y. Molecular characterization of the Waxy locus of rice(Oryza sativa).Plant Cell Physiol. 1991,32:989
78. Hirano HY, Eiguchi M, Sano Y.A single base change altered the regulation of the Wx gene at the posttranscriptional level during the domestrication of rice. Mol Biol Evol, 1998,15:978-987
79. Hirano HY, Sano Y. Comparison of Wx gene regulation in the endosperm and pollen in Oryza sativa L. Genes Genet Syst. 2000, Oct;75(5):245-9
80. Hsieh S C, et al. Evaluation and Genetic Studies on Grain Quality Characters in Rice. Symp. Plant Breed, 1982:99-112
81. Ikeno S. über die Bestaubung und die Bastardierung von Reis. Z Pflanzenzü cht, 1914,2:495-503
82. Isshiki M,Morino K, Nakajima M,et al.A naturally occurring functional allele of the rice Wx locus has a GT to TT mutant at the 5 splice site of the first intron. Plant J, 1998,15:133-321
83. Isshiki M,Nakajima M,Satoh H, et al. Dull rice mutants with tissue-specific effects on the splicing of the Wx pre-Mrna[J]Plant J, 2000,23 (4):451-460
84. ItohK, Ozaki H, Okada K, et al. Introduction of Wx transgene into rice wx mutants leads to both high-and low-amylose rice Plant Cell Physiol. 2003, May;44(5):473-480
85. Jefferson RA. Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep, 1987, 5:387-405
86. Juliano BO. A simplified assay for milled rice amylase. Cereal, 1971, (16):334-340,360
87. Kavi Kishor PB, Reddy GM. Regeneration of rice plants from long-term root and embryo-derived callus culture. Current Sci, 1986,55(14):664-665
88. Khush G. S, Singh R.J,. Sur S. C, et al. Primary trisomics of rice origin, Morphology, cytology and use in linkage mapping. Genetics, 1984,107:141-167
89. Khush G.S. et al. Grain quality consideration in hybrid rice. International
symposium on hybrid Rice. 6-10, October, 1986, Changsha, Hunan, China
90. Khush G.S. Proceedings of the International symposium on hybrid rice. Hybrid Rice, IRRI, Manila. 1988,39-54,201-215
91. K1sgen RJ, Gierl A, Schwarz-Sommer Z, et al. Molecular analysis of the Wx locus of Zea mays. Mol GenGenet, 1986,203:237-244
92. Komari T, Hiei Y, Saito Y, et al. Vectors carrying two separate T-DNAs for cn transformation of higher plants mediated by Agrobacteruim tumefaciens and segregation of transformants free from selection markers. Plant J, 1996, 10(1):165-174
93. Kumar et al. Genetic analysis of different amylose levels in rice. Crop Sci, 1987, (27):1167-1172
94. Kumar I, Khush G S, Juliano BO. Benetic analysis of Wx locus in rice (Oryza sativa L).Theor Appl Genet, 1987,73:481-488
95. Kumria R. Plant regeneration form transformed embryogenie calus of an elite indica rice via Agrobacterium. Plant Cell, Tissus, and Organ Culture, 2001,67:63-67
96. Labra M, Savini C, Bracale M,et al. Genomic changes in transgenie rice(Oryza sativa L.)plants produced by infecting calli with Agrobacterium tumefaciens. Plant Cell Reports. 2001,20:325-330
97. Larkin,P. D., and Park, W.D. Transcript accumulation and utilization of alternate and non-consensus splice sites in rice granule-bound starch synthase are temperature-sensitive and controlled by a single-nucleotide polymorphism. Plant Mol. Biol. 1999,40:719-727
98. Liu Q, Wang Z, Cheng X, et al. Stable inheritance of the antisence Wx gene in transgenic rice with reduced amylose level and improved quality. Transginic Res. 2003Feb;12(1):71-82
99. Martin C and Smith, AM. Starch biosynthesis. Plant Cell. 1995,7:971-985
100. McCormac AC, Fowler MR, Chen DF, et al. Efficient co-transformation of Nicotiana tabacum by two independent T-DNAs, the effect of T-DNA size and implications for genetic separation. Trans Res, 2001, 10:143-155
101. Mikami I, Effects of the two common Wx alleles on different genetic backgrounds in rice. Plant Breed, 2000, 119(6): 505—508
102. Mikami I. Effects of the two common Wx alleles on different genetic backgrounds in rice. Plant Breed, 2000,119(6):505—508
103. Muraue M. The Advance of Rice Taste Evaluation. Tokyo:Japanese Farming News Press, 1997
104. Okagaki RJ,Wessler SR. Comparison of non-mutant and mutant Wx genes in rice and maize. Genetics, 1988,120:1137-1143.
105. Pearson L,Meagher RB. Diverse soybean actin transcripts contain a large intron in the 5 untranslated leader structural similarity to vertebrate muscle actin genes. Plant Mol Biol, 1990,14:513.
106. Pooni H S, et al. A comprehensive model for disomically inherited metrical traits expressed in triploid tissues. Heredity, 1992,62:166-174
107. Puri,R.P.,Siddg, E.A. Inheritance of gelatinization temperature inrice. Ind.J.
Genet. & P1 .Breed, 1980,40(2):450-455
108. Riker, AJ, Lyneis,MM.Locke, S.B. Comparative physiology of crown gall, attenuated crown gall, radiobacter, and hair root bacteria. Phytopathology, 1941,31,964-977
109. Rohde W, Becker D, Salamini F.Structural analysis of the Wx locus from Hordeum vulgare. Nucleic Acids Res 1988, 16:7185—7186
110. Sano Y. Differential regulation of Wx gene expression in rice endosperm. Theor Appl Genet, 1984,68:467-473
111. Sano, Y, Katsumata, M. and Okuno, K. Genetic studies of speciation in cultivated rice. 5. Inter- and intraspecific differentiation in the Wx gene expression of rice. Euphytica, 1985,35:1-9
112. Shimada H, Tada Y, Kawasaki T, et al. Antisense regulation of the rice Wx gene expression using a PCR-amplified fragment of the rice genome reduces the amylase content in grain starch. Theor Appl Genet, 1993,86:665-672
113. Sood B G, Siddiq E A.A rapid technique for scent determination in rice. Crop Sci, 1978,38:268-271
114. Stansel, J.W.The influence of heredity and environment on endosperm characteristics of rice(Oryza sativa L.).Dissertation Abstr. 1996,27:4813
115. Tan Y F. The three important traits for cooking and eating quality of rice grains are controlled by a singlelocus in an elite rice hybrid, shanyou 63. Theoretical and Applied Genetics, 1999, 99(3/4): 642—648
116. Terada R, Nakajima M, Isshiki M, et al. Antisense Wx genes with highly active promoters effectively suppress Wx gene expression in transgenic rice. Plant Cell Physiol. 2000, Jul;41(7):881-8
117. Tomar J B. Genetic Analysis of Amylose Content in Rice. Genetic Agrar, 1987, 41(3): 235—242
118. Vander Krol AR,Mol JN, Stuitje AR. Antisense genes in plants:an overview. Gene,1988 Dec 10,72(1-2):45-50
119. Vasil. V. and Z K, Vasil. Cell culture and somatic cell genetics of plant,In"Induction and Maintenance of Embryogenic callus cultures of cramineae"1984, pp36-42, Academic Press, Orlando, Florida
120. Wang ZY,Zheng FQ, Shen GZ, et al. The amylose content in rice endosperm is related to the post-transcrisptional regulation of the Wx gene. Plant J. 1995 Apr; 7(4):613-22
121. Wang, Z.Y, Zheng, F.Q, Wang, X.Q, et al. Characterization of two transposon like elements in rice Wx gene. Sci. China Series B. 1994,37:437-447
122. Zhou PH, Tan YF, He YQ, et al. Simultaneous improvement for four quality traits of Zhenshan97, an elite parent of hybrid rice,by molecular marker-assisted selection Theor Appl Genet. 2003, Jan;106(2):326-31
123. Zhu Y, Cai XL, Wang ZY, et al. An interaction between a MYC protein and EREBP protein is involved in transcriptional regulation of the rice Wx gene. Biol Chem. 2003,278(48):47803-11