水稻广亲和基因的功能鉴定
摘要
水稻籼粳亚种间具有很强的杂种优势,然而,由于杂种的半不育特性,限制了其优势的进一步利用。值得庆幸的是水稻育种家们发现有些水稻品种与籼稻或粳稻杂交育性均表现正常,这样的品种称为广亲和品种,这一发现为籼粳亚种间杂种优势的利用提供了可能。研究表明控制这一性状的广亲和基因S5n位于水稻第6号染色体上。
本实验室先前的研究将S5n精细定位到分子标记J13和J17之间,物理距离为50 Kb。对此区间的序列进行预测,确定了两个广亲和候选基因,Asp和CG2,分别编码天冬氨酸蛋白酶和一个功能未知的蛋白。对每一个候选基因,我们都构建二类表达载体,即过量表达载体和功能互补测验载体,而后将这些表达载体通过农杆菌介导转入到不含有广亲和基因的受体材料中,检测后代小穗育性,确定广亲和基因。结果如下:
1.直接以‘Balilla’ב南京11’的杂交种为受体获得6个转化系,在海南种植,并检查小穗育性。候选基因Asp的转基因植株与未转任何载体的组培材料相比,小穗育性无明显差异;转基因植株与转空白载体的阴性对照相比,小穗育性明显提高,差异达显著水平,但平均育性仍然很低。
2.以二个粳稻品种,‘Balilla’和‘武香粳’,为受体获得转基因植株,将这些转基因材料与籼稻品种‘南京11’杂交,同时检测杂交后代的基因型和小穗育性。结果表明只有一株转基因‘武香粳’(转Asp过量表达载体)与‘南京11’的杂种后代中,携带候选基因的杂种比同背景的不带候选基因的杂种平均育性提高14.15%,达极显著水平,并且平均育性高于50%。初步结果推测编码天冬氨酸蛋白酶的基因Asp可能与水稻的广亲和特性有关,具体的机理有待进一步研究。
Strong heterosis has been observed in rice inter-subspecific hybrids, however, such heterosis could not find its application in rice production due to an occurrence of semi-sterility spikelets. Fortunately, the rice breeders found some very unique varieties, called wide-compatibility varieties, that could lead to normal fertility in the hybrids when crossing to either indica or japonica rice subspecies. This discovery made it possible to utilize the strong heterosis of inter-subspecific hybrids. The S5n gene, located on chromosome 6, has previously been manifested to be responsible for the wide-compatibility in rice.
In our previous report, the S5n gene has been mapped within a 50 kb interval delimited by the left marker J13 and the right marker J17. Sequence analysis of the 50 kb region revealed two candidate genes, coding an aspartyl protease and a hypothetical protein. In the present study, we constructed the two kinds of express vectors, over-expression vector and functional complementary test vector, for each candidate gene. All the express vectors were transformed into the rice receptors for determining the gene responsible for the wide-compatibility in rice.
The results for transformation are summarized as follows:
1. By using the‘Balilla×Nanjing11’F1 hybrid seeds as receptors, six transgenic lines were obtained and grew in Hainan winter nursery for investigating the spikelet fertilities. Comparing the transgenic lines with the candidate genes with those transformants carrying no vector, no any difference has been observed in the
本实验室先前的研究将S5n精细定位到分子标记J13和J17之间,物理距离为50 Kb。对此区间的序列进行预测,确定了两个广亲和候选基因,Asp和CG2,分别编码天冬氨酸蛋白酶和一个功能未知的蛋白。对每一个候选基因,我们都构建二类表达载体,即过量表达载体和功能互补测验载体,而后将这些表达载体通过农杆菌介导转入到不含有广亲和基因的受体材料中,检测后代小穗育性,确定广亲和基因。结果如下:
1.直接以‘Balilla’ב南京11’的杂交种为受体获得6个转化系,在海南种植,并检查小穗育性。候选基因Asp的转基因植株与未转任何载体的组培材料相比,小穗育性无明显差异;转基因植株与转空白载体的阴性对照相比,小穗育性明显提高,差异达显著水平,但平均育性仍然很低。
2.以二个粳稻品种,‘Balilla’和‘武香粳’,为受体获得转基因植株,将这些转基因材料与籼稻品种‘南京11’杂交,同时检测杂交后代的基因型和小穗育性。结果表明只有一株转基因‘武香粳’(转Asp过量表达载体)与‘南京11’的杂种后代中,携带候选基因的杂种比同背景的不带候选基因的杂种平均育性提高14.15%,达极显著水平,并且平均育性高于50%。初步结果推测编码天冬氨酸蛋白酶的基因Asp可能与水稻的广亲和特性有关,具体的机理有待进一步研究。
Strong heterosis has been observed in rice inter-subspecific hybrids, however, such heterosis could not find its application in rice production due to an occurrence of semi-sterility spikelets. Fortunately, the rice breeders found some very unique varieties, called wide-compatibility varieties, that could lead to normal fertility in the hybrids when crossing to either indica or japonica rice subspecies. This discovery made it possible to utilize the strong heterosis of inter-subspecific hybrids. The S5n gene, located on chromosome 6, has previously been manifested to be responsible for the wide-compatibility in rice.
In our previous report, the S5n gene has been mapped within a 50 kb interval delimited by the left marker J13 and the right marker J17. Sequence analysis of the 50 kb region revealed two candidate genes, coding an aspartyl protease and a hypothetical protein. In the present study, we constructed the two kinds of express vectors, over-expression vector and functional complementary test vector, for each candidate gene. All the express vectors were transformed into the rice receptors for determining the gene responsible for the wide-compatibility in rice.
The results for transformation are summarized as follows:
1. By using the‘Balilla×Nanjing11’F1 hybrid seeds as receptors, six transgenic lines were obtained and grew in Hainan winter nursery for investigating the spikelet fertilities. Comparing the transgenic lines with the candidate genes with those transformants carrying no vector, no any difference has been observed in the
引文
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2. 袁隆平,杂交水稻育种的战略设想,杂交水稻,1987,1:1-3
3. 吴万春,徐雪宾,对稻二亚种命名的意见,中国水稻科学,1988,2(1):36-39
4. 丁颖,中国古来粳籼稻种栽培及分布之探讨与现在栽培稻种分类法预报,中山大学农艺专刊,1949,6:1-32
5. 杨守仁,籼粳杂交育种研究,作物学报,1962,1(2):97—102
6. 曾世雄,杨秀青,卢庄文,栽培稻籼粳亚种间杂种一代优势的研究,作物学报, 1980,6(4):193-202
7. 肖金华,袁隆平,水稻籼粳亚种间杂种一代优势及其与亲本关系的研究,杂交水稻,1998,1:5-9
8. 晏月明,牟致远,籼粳稻亚种间杂种一代优势的研究,四川农业学报,1988,3(2):6-10
9. Kato S, Kosaka H, Hara K (1928) On the affinity of rice varieties as shown by fertility of hybrid plants. Bull Sci Fac Agric Kyushu Univ 3: 132-147
10. 俞履沂,林权,中国栽培稻亲缘关系的研究,作物学报,1962,1: 233-258
11. 杨守仁,沈锡英,顾慰连等,籼粳稻杂交育种研究,遗传学通讯,1973,2:34-38
12. 杜尔滨,H.B 编,植物细胞质雄性不育的遗传学原理,北京:农业出版社,1980:17-18
13. Oka H (1953) The mechanism of sterility in the intervarietal hybrid. Phylogenetic differentiation of the cultivated rice plants. Japan J Breed 2: 217-224
14. Henderon MT (1964) Cytogenetics studies at the Louisiana agricultural experiment station on the nature of intervarieties hybrids sterility in O.sativa. Genetics and cytogenetics 1: 147-153
15. Yao SY, Henderson (1958) Cryptic structural hybridity as a proable cause of sterility in intervarietal hybrids of cultivater rice, Oryza sativa. Cytologia 23: 46-55
16. Ikehashi H, Araki H (1984) Variety screening of compatibility types revealed in F1 fertility of distant cross in rice. Japan J Breed 34:304–313
17. 李晓玲,水稻籼粳亚种间杂种不育性的研究进展,农业生物技术科学,2004,20(5):45-49
18. Kitamura E (1962) Studies on cytoplasmic sterility of hybrids in distantly related varieties of rice (Oryza sativa L.) I. Fertility of the F1 hybrids between strains derived from a certain Philippines x Japanese variety crosses and Japanese varieties. Japan. J. Breed 12(2): 81-84.
19. Ikehashi H (1982) Prospects for overcoming barriers in the utilization of indica-japonica crosses in rice breeding. Oryza 19:69-77
20. Ikehashi H, Wan J (1996) Differentiation of alleles at six loci for hybrid sterility in cultivated rice (Oryza sativa L). In: IRRI, ed. Rice GeneticsⅢ, pp404~408.
21. 张桂权,卢永根,栽培稻杂种不育性的遗传研究Ⅰ. 等基因 F1 不育系杂种不育性的双列分析,中国水稻科学,1989,3(3):97~ 101
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