黄瓜性型遗传规律及性别决定相关基因的分布和表达研究
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摘要
黄瓜(cucumis sativus L．)属葫芦科(Cucurbitaceae)、甜瓜属(Cucumis)、一年生草本蔬菜作物。黄瓜是世界性的重要蔬菜。我国是世界上黄瓜栽培面积最广、总产量最高的国家。雌花节率的高低是影响黄瓜产量的重要因素。同时，利用黄瓜全雌性品系能极大地简化杂交种的制种工作。由于黄瓜性别表现和遗传机制的复杂性，利用传统的方法进行性别调控和雌性的育种效率不高。因此深入研究黄瓜性型的遗传规律，认识黄瓜性别决定相关基因在不同性型黄瓜种质资源中的分布及表达特征，对全面理解黄瓜性别分化的机理，加快雌性品系的选育具有重要的意义，同时可为性别决定基因的分离克隆、体外重组与遗传转化奠定基础。本试验从三个方面进行了研究，得到如下研究结果：
1．利用全雌性系“G5224"、强雌性系“1613EF”分别与普通性型株系“YX05-3-10”杂交、自交和回交，获得了两个组合的F_1、F_2、BC_1P_1、BC_1P_2 6个群体。通过对各组合P_1、P_2、F_1、F_2、BC_1P_1、BC_1P_2六世代植株性型的定性观测，并经χ~2测验，发现全雌和强雌性状分别由一对不完全显性等位基因控制。以两个组合的P_1、P_2、F_1、F_2群体的数据为基础，进一步对性型进行了数量遗传学的分析，表明全雌性性状和强雌性性状均是由一对主基因控制，而且存在微效多基因。在全雌性组合中，主基因的遗传率为83.8％，微效多基因的遗传率为8.5％；在强雌性组合中，主基因的遗传率为82.0％，微效多基因的遗传率为8.6％。
2．利用特异引物PCR方法，检测了黄瓜性别决定相关基因ACC合酶基因CS-ACS1G、ACC氧化酶基因CS-AC02和CS-AC03、乙烯受体基因CS-ETR2和CS-ERS在78份不同性型的黄瓜材料中的分布，发现这5个基因均存在于所有材料的基因组中，不具有性型特异性。还对ACC合酶基因进行了克隆，序列分析证明扩增产物正是ACC合酶基因CS-ACS1G。说明多数学者认为的CS-ACS1G具有雌性特异性的结论不具有广泛的适用性。
3．利用半定量RT-PCR方法研究了ACC合酶基因CS-ACS1G、ACC氧化酶基因CS-ACO2和CS-AC03、乙烯受体基因CS-ETR2和CS-ERS在全雌性材料“G5224”、强雌性材料“1613EF”和“106BE”、普通性型材料“YX05-47-4”和西双版纳黄瓜“BN32'’经或不经性别诱导处理后8个时间段的表达特征。结果发现，就每一个基因在不同性型材料中的表达来看，ACC合酶基因CS-ACS1G在所有样品中均未表达，说明该基因在苗期的自然表达与否，与后期植株的性型表现没有直接的关系，与前人的研究结果基本一致。ACC氧化酶基因大多表现瞬时或间歇性表达，且仅在全雌性材料和普通性型材料中，表现了与雌性的一定程度的协同关系。乙烯受体基因CS-ERS仅在雌性材料“G5224”和强雌性材料“106BE”中瞬间表达，硝酸银明显抑制了其表达，而在普通性型材料中均未表达，表现了与雌性材料的特异协同关系；乙烯受体基因CS-ETR2在所有样品中均有表达，且硝酸银能抑制其表达(除“1613EF”外)，乙烯利促进其表达，其在5个材料中的表达与雌性的表现有一定的相关性。就所有基因在同一材料中的表达来看，在全雌性黄瓜材料中，ACC氧化酶基因CS-ACO2和CS-ACO3、乙烯受体基因CS-ETR2和CS-ERS 4个雌性相关基因得到了表达，并且均表现了与雌性的相关性。在强雌材料“1613EF”中，CS-ACO2、CS-ACO3和CS-ETR2有表达，但两个ACC氧化酶基因的表达在硝酸银处理后反而被促进，乙烯受体基因CS-ETR2则在处理和对照中的表达无差异。在强雌材料“106BE”中，4个基因均有表达，两个ACC氧化酶基因的表达与在“1613EF”中的表达有相似之处，两个乙烯受体基因的表达则表现出与雌性的协同关系。在普通性型材料“YX05-47-4”中，除CS-ERS外的ACC氧化酶基因和乙烯受体基因均有表达，且与雌性表现存在一定的协同关系。在西双版纳黄瓜中，仅有CS-ACO3、CS-ETR2 2个雌性相关基因表达，并表现出与雌性的协同关系。可见，每个性别决定基因在植株的性别表现中并非具有同等效应和同样的协同关系；不同性型材料的性别表现可能受不同性别决定基因的表达消长和调控。
Cucumber (Cucumis sativus L.) is the annual herbaceous crops. Cucumber is one of the important vegetable crops in the world. China has the most area of cucumber cultivation and the highest production in the world. Female flower node proportion is a major factor affecting cucumber yield. Meanwhile, the utilization of gynoecious cucumber lines can greatly simplify the Hybridization. As the complexity of cucumber sex expression and genetic mechanism, the traditional methods of sex control and the gynoecious line breeding are not efficient. Therefore, the further study on the genetic dissection of sex expression and the distribution and expression characteristics of genes related to sex determination in Cucumber(Cucumis sativus L.) is important to fully understand the mechanism of sex formation and accelerate the breeding speed. In addition, it could lay the foundation for the isolation and cloning, in vitro reorganization and genetic transformation of genes on sex determination. The researches was done from three aspects and the results are as follows:
1. Two sets of F_2, BC_1P_1, BC_1P_2 population were respectively constructed from the cross combination of gynoecious line 'G5224' and monoecious line 'YX05-3-10', and subgynoecious line '1613EF' and 'YX05-3-10'. Through investigation andχ~2 test of sex types in segregating population, it was found that both gynoecious and subgynoecious traits were controlled by one pair of incompletely dominant gene. With the help of the major gene and polygene mixed inheritance model from the aspect of quantitative genetics, It was proved that there were polygenes involved in both gynoecious and subgynoecious inheritance. The heritability of major gene of the cross of gynoecious line 'G5224' and monoecious line 'YX05-3-10' was 83.8%, and the heritability of polygenes was 8.5%. The heritability of major gene of the cross of subgynoecious line '1613EF' and monoecious cucumber 'YX05-3-10' was 82.0%, and the heritability of polygenes was 8.6%.
2. The distribution of the genes related to sex determination in 78 cucumber germplasm materials of different sex pheynotype was analyzed by specific PCR primers based on the sequences of five genes: CS-ACS1G, CS-AC02, CS-AC03, CS-ERS and CS-ETR2. The results showed that all the 5 genes could be detected in all the cucumber materials. It meant that the five genes were not specific to the gynoecius genotypes. ACC synthase gene was cloned from gynoecious line 'G5224'. BLAST analysis revealed that the sequence was highly homologous to the sequence of CS-ACSIG gene at 99%. The results from some other researchers that CS-ACS1G gene existed only in gynoecious cucumbers were not suitable for cucumber germplasm resources with widely genetic beckground.
3. The technique of semi-quantitative RT-PCR was employed to study the expression character of ACC synthase gene CS-ACS1G, ACC oxidase gene CS-AC02, CS-AC03 and ethylene receptor gene CS-ERS and CS-ETR2 at 8 different time after treatment on gynoecious line 'G5224', subgynoecious line '1613EF' and '106BE', monoecious line 'YX05-3-10' and 'Xi Shuang Ban Na cucumber 'BN32'. The results showed that ACC synthase gene CS-ACSIG didn't express in all the 80 samples, which showed that the sex pheynotype at late stage of plant growth was not directly related to whether this gene expressed naturally or not in the early seedling stage. The expression of ACC oxidase genes was mostly transient or intermittent only in gynoecious and monoecious cucumber lines, whichshowed synergies with the gynoecious phenotype. But in the subgynoecious line which was strongly reactive to AgNO3, the expression of the ACC oxidase gene was enhanced. This may be associated with different background and different expression machanisms in different sexual materials. Ethylene receptor gene CS-ERS only showed transient expresstion in the gynoecious cucumber 'G5224' and subgynoecious cucumber '106BE'. AgNO3 significantly inhibited its expression. All these showed Ethylene receptor gene CS-ERS has female-specific relation with the gynoecious trait. Ethylene receptor gene CS-ETR2 expressed in all 80 samples to differnt extent. AgNO3 could inhibit the expression (except '1613EF'), and ethylene could promote its expression. Its expression in 5 materials showed higher relative to the gynoecious trait.
Viewing on the genes existed in one material, there were 4 genes expressed in gynoecious line 'G5224', including 2 ACC oxidase genes and 2 ethylene receptor genes, and all of them showed some relation with gynoecious trait. In subgynoecious line '1613EF', CS-ACO2,CS-AC03 and CS-ETR2 genes expressed. In subgynoecious line '106BE', all the 2 ACC oxidase genes and 2 ethylene receptor genes expressed. The change of ACC oxidase genes in the 2 subgynoecious lines showed abnormal relation with sex type, and ethylene receptor genes showed synergies with the gynoecious trait. In the monoecious line 'YX05-3-10', CS-ACO2,CS-AC03 and CS-ETR2 expressed, and all of them showed synergies with the gynoecious plant. In the monoecious cucumbe 'Xi Shuang Ban Na cucumber (BN32)', only CS-AC03 and CS-ETR2 expressed, and their expression was synergic with gynoecious. It could be inferred that each of the 4 sex determination genes mentioned above couldn't has the same effect on and the same synergies with the gynoecious performance; Ethylene receptor gene seemed to play more important role in sex-determination; Different sex-determining genes may be responsible for the different sex type. That is, The phenotypes of different sex may be controlled by the different sex-determining genes.
1．利用全雌性系“G5224"、强雌性系“1613EF”分别与普通性型株系“YX05-3-10”杂交、自交和回交，获得了两个组合的F_1、F_2、BC_1P_1、BC_1P_2 6个群体。通过对各组合P_1、P_2、F_1、F_2、BC_1P_1、BC_1P_2六世代植株性型的定性观测，并经χ~2测验，发现全雌和强雌性状分别由一对不完全显性等位基因控制。以两个组合的P_1、P_2、F_1、F_2群体的数据为基础，进一步对性型进行了数量遗传学的分析，表明全雌性性状和强雌性性状均是由一对主基因控制，而且存在微效多基因。在全雌性组合中，主基因的遗传率为83.8％，微效多基因的遗传率为8.5％；在强雌性组合中，主基因的遗传率为82.0％，微效多基因的遗传率为8.6％。
2．利用特异引物PCR方法，检测了黄瓜性别决定相关基因ACC合酶基因CS-ACS1G、ACC氧化酶基因CS-AC02和CS-AC03、乙烯受体基因CS-ETR2和CS-ERS在78份不同性型的黄瓜材料中的分布，发现这5个基因均存在于所有材料的基因组中，不具有性型特异性。还对ACC合酶基因进行了克隆，序列分析证明扩增产物正是ACC合酶基因CS-ACS1G。说明多数学者认为的CS-ACS1G具有雌性特异性的结论不具有广泛的适用性。
3．利用半定量RT-PCR方法研究了ACC合酶基因CS-ACS1G、ACC氧化酶基因CS-ACO2和CS-AC03、乙烯受体基因CS-ETR2和CS-ERS在全雌性材料“G5224”、强雌性材料“1613EF”和“106BE”、普通性型材料“YX05-47-4”和西双版纳黄瓜“BN32'’经或不经性别诱导处理后8个时间段的表达特征。结果发现，就每一个基因在不同性型材料中的表达来看，ACC合酶基因CS-ACS1G在所有样品中均未表达，说明该基因在苗期的自然表达与否，与后期植株的性型表现没有直接的关系，与前人的研究结果基本一致。ACC氧化酶基因大多表现瞬时或间歇性表达，且仅在全雌性材料和普通性型材料中，表现了与雌性的一定程度的协同关系。乙烯受体基因CS-ERS仅在雌性材料“G5224”和强雌性材料“106BE”中瞬间表达，硝酸银明显抑制了其表达，而在普通性型材料中均未表达，表现了与雌性材料的特异协同关系；乙烯受体基因CS-ETR2在所有样品中均有表达，且硝酸银能抑制其表达(除“1613EF”外)，乙烯利促进其表达，其在5个材料中的表达与雌性的表现有一定的相关性。就所有基因在同一材料中的表达来看，在全雌性黄瓜材料中，ACC氧化酶基因CS-ACO2和CS-ACO3、乙烯受体基因CS-ETR2和CS-ERS 4个雌性相关基因得到了表达，并且均表现了与雌性的相关性。在强雌材料“1613EF”中，CS-ACO2、CS-ACO3和CS-ETR2有表达，但两个ACC氧化酶基因的表达在硝酸银处理后反而被促进，乙烯受体基因CS-ETR2则在处理和对照中的表达无差异。在强雌材料“106BE”中，4个基因均有表达，两个ACC氧化酶基因的表达与在“1613EF”中的表达有相似之处，两个乙烯受体基因的表达则表现出与雌性的协同关系。在普通性型材料“YX05-47-4”中，除CS-ERS外的ACC氧化酶基因和乙烯受体基因均有表达，且与雌性表现存在一定的协同关系。在西双版纳黄瓜中，仅有CS-ACO3、CS-ETR2 2个雌性相关基因表达，并表现出与雌性的协同关系。可见，每个性别决定基因在植株的性别表现中并非具有同等效应和同样的协同关系；不同性型材料的性别表现可能受不同性别决定基因的表达消长和调控。
Cucumber (Cucumis sativus L.) is the annual herbaceous crops. Cucumber is one of the important vegetable crops in the world. China has the most area of cucumber cultivation and the highest production in the world. Female flower node proportion is a major factor affecting cucumber yield. Meanwhile, the utilization of gynoecious cucumber lines can greatly simplify the Hybridization. As the complexity of cucumber sex expression and genetic mechanism, the traditional methods of sex control and the gynoecious line breeding are not efficient. Therefore, the further study on the genetic dissection of sex expression and the distribution and expression characteristics of genes related to sex determination in Cucumber(Cucumis sativus L.) is important to fully understand the mechanism of sex formation and accelerate the breeding speed. In addition, it could lay the foundation for the isolation and cloning, in vitro reorganization and genetic transformation of genes on sex determination. The researches was done from three aspects and the results are as follows:
1. Two sets of F_2, BC_1P_1, BC_1P_2 population were respectively constructed from the cross combination of gynoecious line 'G5224' and monoecious line 'YX05-3-10', and subgynoecious line '1613EF' and 'YX05-3-10'. Through investigation andχ~2 test of sex types in segregating population, it was found that both gynoecious and subgynoecious traits were controlled by one pair of incompletely dominant gene. With the help of the major gene and polygene mixed inheritance model from the aspect of quantitative genetics, It was proved that there were polygenes involved in both gynoecious and subgynoecious inheritance. The heritability of major gene of the cross of gynoecious line 'G5224' and monoecious line 'YX05-3-10' was 83.8%, and the heritability of polygenes was 8.5%. The heritability of major gene of the cross of subgynoecious line '1613EF' and monoecious cucumber 'YX05-3-10' was 82.0%, and the heritability of polygenes was 8.6%.
2. The distribution of the genes related to sex determination in 78 cucumber germplasm materials of different sex pheynotype was analyzed by specific PCR primers based on the sequences of five genes: CS-ACS1G, CS-AC02, CS-AC03, CS-ERS and CS-ETR2. The results showed that all the 5 genes could be detected in all the cucumber materials. It meant that the five genes were not specific to the gynoecius genotypes. ACC synthase gene was cloned from gynoecious line 'G5224'. BLAST analysis revealed that the sequence was highly homologous to the sequence of CS-ACSIG gene at 99%. The results from some other researchers that CS-ACS1G gene existed only in gynoecious cucumbers were not suitable for cucumber germplasm resources with widely genetic beckground.
3. The technique of semi-quantitative RT-PCR was employed to study the expression character of ACC synthase gene CS-ACS1G, ACC oxidase gene CS-AC02, CS-AC03 and ethylene receptor gene CS-ERS and CS-ETR2 at 8 different time after treatment on gynoecious line 'G5224', subgynoecious line '1613EF' and '106BE', monoecious line 'YX05-3-10' and 'Xi Shuang Ban Na cucumber 'BN32'. The results showed that ACC synthase gene CS-ACSIG didn't express in all the 80 samples, which showed that the sex pheynotype at late stage of plant growth was not directly related to whether this gene expressed naturally or not in the early seedling stage. The expression of ACC oxidase genes was mostly transient or intermittent only in gynoecious and monoecious cucumber lines, whichshowed synergies with the gynoecious phenotype. But in the subgynoecious line which was strongly reactive to AgNO3, the expression of the ACC oxidase gene was enhanced. This may be associated with different background and different expression machanisms in different sexual materials. Ethylene receptor gene CS-ERS only showed transient expresstion in the gynoecious cucumber 'G5224' and subgynoecious cucumber '106BE'. AgNO3 significantly inhibited its expression. All these showed Ethylene receptor gene CS-ERS has female-specific relation with the gynoecious trait. Ethylene receptor gene CS-ETR2 expressed in all 80 samples to differnt extent. AgNO3 could inhibit the expression (except '1613EF'), and ethylene could promote its expression. Its expression in 5 materials showed higher relative to the gynoecious trait.
Viewing on the genes existed in one material, there were 4 genes expressed in gynoecious line 'G5224', including 2 ACC oxidase genes and 2 ethylene receptor genes, and all of them showed some relation with gynoecious trait. In subgynoecious line '1613EF', CS-ACO2,CS-AC03 and CS-ETR2 genes expressed. In subgynoecious line '106BE', all the 2 ACC oxidase genes and 2 ethylene receptor genes expressed. The change of ACC oxidase genes in the 2 subgynoecious lines showed abnormal relation with sex type, and ethylene receptor genes showed synergies with the gynoecious trait. In the monoecious line 'YX05-3-10', CS-ACO2,CS-AC03 and CS-ETR2 expressed, and all of them showed synergies with the gynoecious plant. In the monoecious cucumbe 'Xi Shuang Ban Na cucumber (BN32)', only CS-AC03 and CS-ETR2 expressed, and their expression was synergic with gynoecious. It could be inferred that each of the 4 sex determination genes mentioned above couldn't has the same effect on and the same synergies with the gynoecious performance; Ethylene receptor gene seemed to play more important role in sex-determination; Different sex-determining genes may be responsible for the different sex type. That is, The phenotypes of different sex may be controlled by the different sex-determining genes.
引文
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19．娄群峰．黄瓜全雌性基因分子标记及ACC合酶基因克隆与表达研究．南京农业大学二零零四届博士学位论文
20．娄群峰，陈劲枫，Mouy Jahn，陈龙正，耿红，罗向东．黄瓜全雌性基因连锁的AFLP和SCAR分子标记．园艺学报，2005，32(2)：256-261
21．罗宗洛．植物生理知识，科学出版社，973，101-102
22．孟繁静．植物花发育的分子生物学．2000．中国农业出版社
23．米宁娜(苏)．在外界环境影响下植物性别的转变．孙岱译．北京：科学出版社，1957
24．任吉君，王艳．黄瓜性别决定解剖学研究，北方园艺，1994 46-47
25．寿森炎，汪俏梅．高等植物性别分化研究进展．植物学通报，2000，17(6)：528-535
26．孙晓红．草菇冷诱导相关基因的克隆与分析．南京农业大学二零零六年博士学位论文
27．谭其猛．蔬菜育种．北京：农业出版社，1980
28．汪俏梅，曾广文．苦瓜性别分化特异蛋白质的研究．植物学报，1998，40(3)：241-246
29．王建康，盖钧镒．利用杂种F_2世代鉴定数量性状主基因-多基因混合遗传模型并估计其遗传效应．植物学报，24(5)：432-440，1997
30．王彦华，侯喜林，申书兴．不结球白菜β-1，3-葡聚糖酶基因cDNA全长的克隆与表达分析．农业生物技术学报，2006，14(6)：917～921
31．吴乃虎．基因工程原理(第二版)．北京：科学出版社，1998：100
32．吴叶青．乙烯利在夏黄瓜上的应用效果．长江蔬菜．2000，(12)：37-38
33．夏仁学．园艺植物性别分化的研究进展，植物学通报，1996，13(增刊)：12-19
34．杨永华，盖钧镒，马育华．春夏秋播种季节条件下大豆生育期遗传的差异表现．中国农业科学，1994，27(3)：1-6
35．叶波平，吉成均，杨玲玲，等．不同性别表型黄瓜基因组中雌性系特异的ACC合酶基因[J]．植物学报2000，42(20)；164-168
36．应振土，李曙轩．瓜类性别表现的研究进展．生物科学动态，1989，5(2)：6-9
37．应振土，李曙轩．瓠瓜与黄瓜的性别表达和内源乙烯与氧化酶活性的关系．园艺学报，1990，17 (1)：51-57
38．于凤鸣，刘玉艳，杨越冬．乙烯利诱导黄瓜雌花分化机理初探(简报)，河北农业技术示范学院学报，1997，11(2)：68-70
39．斋藤隆．Sex differentiation of cucubita(Ⅰ)[M]，见《蔬菜译丛》，Beijing：Agricultural Press 1982．p：39-42(in Chinese)
40．周建平，杨足君，冯娟等．小麦蛋白翻译起始因子5A基因(elF5A)的克隆与分析．遗传，2006，28(5)：571～577
41. Ando S, Sato Y, Kamachi S, Sakai S.Isolation of a MADS-box gene (ERAF17) and correlation of its expression with the induction of formation of female flowers by ethylene in cucumber plants (Cucumis sativus L.)[J]. Planta. 2001, Oct; 213(6): 943-952
42. Atsmon D. Galun E. A morphogenetic study of staminate, pistillate and hermaphrodite flowers in Cucumis Sativus (L.) (J) Phytomorphology, 1960,10: 110-115
43. Atsmon D, Tabbak C. Comparative effects of gibberellin, silver nitrate, and aminoethoxyvinyl glycine on sexual tendency and ethylene evolution in the cucumber plant (Cucumis sativus L.). Plant Cell Physiol, 1979, 20: 1547-1555
44. Bauer D, Warthoe P, Rohde M, et al. Detection and differential display of expression genes by DDRT-PCR. PCR Methods Appli, 1994, 4: S97-108.
45. Barker H, Solomon-Blackburn R. M, Mcnicol J. W., Bradshaw J. E. Resistance to potato leaf roll virus multiplication in potato is under major gene control. Theor. Appl. Genet., 1994a., 88: 754-758.
46. Beyer E. Silver ion: a potent antiethylene agent in cucumber and tomato. Hort. Sci., 1976, 11:195-196
47. Conner J. Tests for major genes affecting quantitative traits in wild radish Raphanus Raphanistrum.Genetica, 1993,90:41-45
48. Cross NCR Quantitative PCR techniques and applications.Br J Haematol ,1995 ,89 :693
49. Galun E. The role of auxins in the sex expression of the cucumber. Physiol. Plant, 1959,12:48-61
50. Galun E. Study of the inheritance of sex expression in the cucumber--The interaction of major genes with modifying genetic and non-genetic factors. Genetica, 1961, 32: 134-163
51. Galun E, Jung Y, Lang. A culture and sex modification of male cucumberbuds in vitro.Nature, 1962,194:596
52. Gretch D , Corey L, Wilson J , et al. Assessment of hepatitis C virus RNA levels by quantitative competitive RNA polymerase chain reaction : high - titer viremia correlates with advanced stage of disease. J Infect Dis , 1994 ,169 :1219 - 1225
53. Goffinet MC. 1990. Comparative ontogeny of male and female flowers of Cucumis sativus.In: Bates DM, Robinson RW, Jeffrey C (eds) Biology and utilization of the Cucurbitaceae. Cornell University Press, New York, pp 288 - 304
54. Gorge WL. Influence of genetic background on sex conversion by 2-chloroethylphosphonic acid in monoecious cucumbers. J. Amer. Soc. Hort. Sci,1971,96: 152-154
55. Hagemann LJ et al. Development of bovine embryos in single in vitro production (sIVP) systems. Theriogenology, 1997 ,45 :254.
56. Hoeschele I. Statistical techniques for detection of major genes in animal breeding data Theor. Appl. Genet, 1988, 76: 311-319
57. Iwahori S, Lyons JM, Smith OE. Sex expression in cucumber plants as affected by 2-chloroethanephosphonic acid, ehylene, and growth regulators. Plant Physiol,1970,46:412-415
58. Jalava T, Lehtovaara P , Kallio A ,et al. Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates. Biotechniques. 1993 ,15 : 134 - 139
59. Kahana A, Silberstein L, Kessler N, Goldstein RS, Perl-Treves R.Expression of ACC oxidase genes differs among sex genotypes and sex phases in cucumber [J]. Plant Mol Biol. 1999 Nov; 41(4): 517-528
60. Kamachi S, Sekimoto H, Kondo N, Sakai S.Cloning of a cDNA for a 1- aminocyclopropane-1-carboxylate synthase that is expressed during development of female flowers at the apices of Cucumis sativus [J] LPlant Cell Physiol. 1997 Nov; 38(11): 1197-1206
61. Kaneko S , Murakami S , Unoura M, Kobayashi K. Quantitation of hepatitis C virus RNA by competitive polymerase chain reaction. J Med Virol, 1992 ,37 : 278 - 282
62. Kreike CM, Dekoning JRA, Vinke JH, van Ooijen JW, Stiekema WJ. Quantitatively-inherited resistance to Globodera pallida is dominated by one major locus in Solanum spegazzinii. Theor Appl Genet, 1994, 88:764-769
2．C．W迪芬巴赫，GS．德维克斯勒．PCR技术实验指南[M]．北京：科学出版社，1998：205-20
3．陈劲枫，娄群峰，俞纪柱，庄飞云．黄瓜性别连锁的分子标记筛选．上海农业学报，2003，19(4)：11-14
4．陈惠明，刘晓虹．黄瓜性型遗传规律的研究．湖南农业大学学报，1999，25(1)：40—43
5．陈惠明，卢向阳等．两个新发现的黄瓜性别决定基因遗传规律的研究．园艺学报，2005，32(5)：895-898
6．陈惠明，许亮，卢向阳，易克，许勇，张海英，刘晓虹．黄瓜雌性性状主控基因CsACSlG的分析及其定位．分子植物育种，2005，3(4)：520-524．
7．陈学好，曾广文，陈艳萍，曹碚生．植物激素和多胺与黄瓜性别逆转的关系．浙江大学学报：农业与生命科学版，2001，27(6)：639-b42
8．陈学好，曹广文，曹碚生．黄瓜花性别分化和内源激素的关系．植物生理学通讯，2002，38(4)：317-320
9．程立宝，秦智伟，李淑艳，丁国华，周秀艳．与黄瓜雌性性状连锁的cs-acslg基因特异片段克隆与鉴定．中国农业科学，2006，39(12)：2545-2550．
10．迟志宏，郑文尧，梁浩．人胃癌组织中生长抑素2型受体表达的初步研究[[J]．中国肿瘤临床与康复，2001．8(2)：18-20
11．杜盈，姚远，刘进元．两个水稻GDP-甘露糖-3'，5'-异构酶基因特征及表达模式的研究(英文)．生物化学与生物物理进展，2006，33(4)：368～376
12．冯志勇．香菇低温胁迫下的生理变化及其应答基因的分离[D]．南京农业大学博士学位论文，2004
13．盖均镒，章元明，王建康。植物数量性状遗传体系．北京：科学出版社，2003
14．黄伟波．外界环境对长春密刺黄瓜性型分化与产量的影响．中国蔬菜．1996，(4)：26-27
15．K B穆里斯，F费里，R吉布斯等．PCR聚合酶链式反应[M]．北京：科学出版社，1997：9
16．李怀智．我国黄瓜栽培的现状及其发展趋势．蔬菜，2003，(8)：3-4
17．李利敏．黄瓜性型分化的内外因素及控制．长江蔬菜．1993，(6)：35
18．李曙轩，傅炳通．黄瓜及瓠瓜的性别表现与激素控制．植物生理学报，1979，510：83-92
19．娄群峰．黄瓜全雌性基因分子标记及ACC合酶基因克隆与表达研究．南京农业大学二零零四届博士学位论文
20．娄群峰，陈劲枫，Mouy Jahn，陈龙正，耿红，罗向东．黄瓜全雌性基因连锁的AFLP和SCAR分子标记．园艺学报，2005，32(2)：256-261
21．罗宗洛．植物生理知识，科学出版社，973，101-102
22．孟繁静．植物花发育的分子生物学．2000．中国农业出版社
23．米宁娜(苏)．在外界环境影响下植物性别的转变．孙岱译．北京：科学出版社，1957
24．任吉君，王艳．黄瓜性别决定解剖学研究，北方园艺，1994 46-47
25．寿森炎，汪俏梅．高等植物性别分化研究进展．植物学通报，2000，17(6)：528-535
26．孙晓红．草菇冷诱导相关基因的克隆与分析．南京农业大学二零零六年博士学位论文
27．谭其猛．蔬菜育种．北京：农业出版社，1980
28．汪俏梅，曾广文．苦瓜性别分化特异蛋白质的研究．植物学报，1998，40(3)：241-246
29．王建康，盖钧镒．利用杂种F_2世代鉴定数量性状主基因-多基因混合遗传模型并估计其遗传效应．植物学报，24(5)：432-440，1997
30．王彦华，侯喜林，申书兴．不结球白菜β-1，3-葡聚糖酶基因cDNA全长的克隆与表达分析．农业生物技术学报，2006，14(6)：917～921
31．吴乃虎．基因工程原理(第二版)．北京：科学出版社，1998：100
32．吴叶青．乙烯利在夏黄瓜上的应用效果．长江蔬菜．2000，(12)：37-38
33．夏仁学．园艺植物性别分化的研究进展，植物学通报，1996，13(增刊)：12-19
34．杨永华，盖钧镒，马育华．春夏秋播种季节条件下大豆生育期遗传的差异表现．中国农业科学，1994，27(3)：1-6
35．叶波平，吉成均，杨玲玲，等．不同性别表型黄瓜基因组中雌性系特异的ACC合酶基因[J]．植物学报2000，42(20)；164-168
36．应振土，李曙轩．瓜类性别表现的研究进展．生物科学动态，1989，5(2)：6-9
37．应振土，李曙轩．瓠瓜与黄瓜的性别表达和内源乙烯与氧化酶活性的关系．园艺学报，1990，17 (1)：51-57
38．于凤鸣，刘玉艳，杨越冬．乙烯利诱导黄瓜雌花分化机理初探(简报)，河北农业技术示范学院学报，1997，11(2)：68-70
39．斋藤隆．Sex differentiation of cucubita(Ⅰ)[M]，见《蔬菜译丛》，Beijing：Agricultural Press 1982．p：39-42(in Chinese)
40．周建平，杨足君，冯娟等．小麦蛋白翻译起始因子5A基因(elF5A)的克隆与分析．遗传，2006，28(5)：571～577
41. Ando S, Sato Y, Kamachi S, Sakai S.Isolation of a MADS-box gene (ERAF17) and correlation of its expression with the induction of formation of female flowers by ethylene in cucumber plants (Cucumis sativus L.)[J]. Planta. 2001, Oct; 213(6): 943-952
42. Atsmon D. Galun E. A morphogenetic study of staminate, pistillate and hermaphrodite flowers in Cucumis Sativus (L.) (J) Phytomorphology, 1960,10: 110-115
43. Atsmon D, Tabbak C. Comparative effects of gibberellin, silver nitrate, and aminoethoxyvinyl glycine on sexual tendency and ethylene evolution in the cucumber plant (Cucumis sativus L.). Plant Cell Physiol, 1979, 20: 1547-1555
44. Bauer D, Warthoe P, Rohde M, et al. Detection and differential display of expression genes by DDRT-PCR. PCR Methods Appli, 1994, 4: S97-108.
45. Barker H, Solomon-Blackburn R. M, Mcnicol J. W., Bradshaw J. E. Resistance to potato leaf roll virus multiplication in potato is under major gene control. Theor. Appl. Genet., 1994a., 88: 754-758.
46. Beyer E. Silver ion: a potent antiethylene agent in cucumber and tomato. Hort. Sci., 1976, 11:195-196
47. Conner J. Tests for major genes affecting quantitative traits in wild radish Raphanus Raphanistrum.Genetica, 1993,90:41-45
48. Cross NCR Quantitative PCR techniques and applications.Br J Haematol ,1995 ,89 :693
49. Galun E. The role of auxins in the sex expression of the cucumber. Physiol. Plant, 1959,12:48-61
50. Galun E. Study of the inheritance of sex expression in the cucumber--The interaction of major genes with modifying genetic and non-genetic factors. Genetica, 1961, 32: 134-163
51. Galun E, Jung Y, Lang. A culture and sex modification of male cucumberbuds in vitro.Nature, 1962,194:596
52. Gretch D , Corey L, Wilson J , et al. Assessment of hepatitis C virus RNA levels by quantitative competitive RNA polymerase chain reaction : high - titer viremia correlates with advanced stage of disease. J Infect Dis , 1994 ,169 :1219 - 1225
53. Goffinet MC. 1990. Comparative ontogeny of male and female flowers of Cucumis sativus.In: Bates DM, Robinson RW, Jeffrey C (eds) Biology and utilization of the Cucurbitaceae. Cornell University Press, New York, pp 288 - 304
54. Gorge WL. Influence of genetic background on sex conversion by 2-chloroethylphosphonic acid in monoecious cucumbers. J. Amer. Soc. Hort. Sci,1971,96: 152-154
55. Hagemann LJ et al. Development of bovine embryos in single in vitro production (sIVP) systems. Theriogenology, 1997 ,45 :254.
56. Hoeschele I. Statistical techniques for detection of major genes in animal breeding data Theor. Appl. Genet, 1988, 76: 311-319
57. Iwahori S, Lyons JM, Smith OE. Sex expression in cucumber plants as affected by 2-chloroethanephosphonic acid, ehylene, and growth regulators. Plant Physiol,1970,46:412-415
58. Jalava T, Lehtovaara P , Kallio A ,et al. Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates. Biotechniques. 1993 ,15 : 134 - 139
59. Kahana A, Silberstein L, Kessler N, Goldstein RS, Perl-Treves R.Expression of ACC oxidase genes differs among sex genotypes and sex phases in cucumber [J]. Plant Mol Biol. 1999 Nov; 41(4): 517-528
60. Kamachi S, Sekimoto H, Kondo N, Sakai S.Cloning of a cDNA for a 1- aminocyclopropane-1-carboxylate synthase that is expressed during development of female flowers at the apices of Cucumis sativus [J] LPlant Cell Physiol. 1997 Nov; 38(11): 1197-1206
61. Kaneko S , Murakami S , Unoura M, Kobayashi K. Quantitation of hepatitis C virus RNA by competitive polymerase chain reaction. J Med Virol, 1992 ,37 : 278 - 282
62. Kreike CM, Dekoning JRA, Vinke JH, van Ooijen JW, Stiekema WJ. Quantitatively-inherited resistance to Globodera pallida is dominated by one major locus in Solanum spegazzinii. Theor Appl Genet, 1994, 88:764-769
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