水稻黄绿叶突变体ygl11(t)的生理特性和基因克隆

doi:10.3969/j.issn.1001-7216.2015.02.001

摘要/Abstract

摘要：

在水稻品种南粳41中发现了一个黄绿叶自然突变体,经过多代连续自交形成了稳定的突变系,命名为ygl11(t),ygl11(t)整个生育期叶片都表现为黄绿色。对苗期、分蘖盛期、齐穗期突变体和野生型的叶绿素含量进行测定,ygl11(t) 的叶绿素含量是野生型的45.7%~74.7%,叶绿素a 含量是野生型的55.2%~87.5%,叶绿素b含量是野生型的12.5%~25.3%,ygl11(t) 的类胡萝卜素的含量是野生型的62.3%~97.0%。ygl11(t)在分蘖盛期的净光合速率显著高于野生型,花后10 d,ygl11(t)的净光合速率比野生型略低。对突变体叶片中叶绿体的超微结构进行观察,发现突变体叶绿体内的类囊体基粒片层数目减少且严重扭曲变形。遗传分析表明,ygl11(t)叶色性状受1对隐性核基因控制。利用SSR分子标记将YGL11(t)初步定位在水稻第10染色体的长臂上,进一步利用新开发的InDel和CAPS标记将YGL11(t)定位在58.1 kb的物理距离内。对该区段内存在的开放阅读框进行序列分析,发现突变体ygl11(t) 中编码叶绿素a氧化酶(chlorophyll a oxygenase 1)基因(OsCAO 1) 的第9个外显子存在2个碱基缺失,从而导致提前出现终止密码子,初步分析OsCAO1即为YGL11(t)的候选基因。

关键词: 水稻, 黄绿叶突变体, 精细定位, 基因, OsCAO1

Abstract:

A spontaneous mutant showing yellow-green leaf throughout the growth period, designated as ygl11(t), was screened and identified from rice cultivar Nanjing 41. This mutant was grown for several generations and showed stable mutation phenotype. The ygl11(t) mutant has reduced chlorophyll accumulation and abnormal chloroplast development. The average contents of total chlorophyll, chlorophyll a, chlorophyll b, and carotene in the ygl11(t) mutant were 45.7% to 74.7%, 55.2% to 87.5%, 12.5% to 25.3%, and 62.3% to 97.0% of these in wild type, respectively, at the stages of seedling, tillering and heading. The leaf net photosynthetic rate of the ygl11(t) was significantly higher at maximum tillering stage and slightly lower at 10 days after flowering than that of wild type. Ultrastructure analysis revealed that the grana lamellae of chloroplast thylakoid were less in number and abnormally developed in the ygl11(t) mutant. Genetic analysis suggested that the phenotype of ygl11(t) was controlled by a recessive nuclear gene. YGL11(t)was prelimarily mapped on the long arm of rice chromosome 10, and was finally narrowed to a 58.1 kb region containing six open reading frames (ORFs). All the six ORFs were amplified and sequenced. A 2-bp deletion was found in the 9th exon of the OsCAO1gene, encoding a chlorophyll a oxygenase 1, which results in a premature stop codon and protein truncation in the ygl11(t) mutant. These results suggest that the OsCAO1 gene is the candidate of YGL11(t).

Key words: rice, yellow-green leaf mutant, fine mapping, gene, OsCAO1

中图分类号:

Q343.5
Q785

吴书俊, 杨杰, 闫影, 张丽霞, 范方军, 朱金燕, 李文奇, 仲维功, 曹黎明, 王军. 水稻黄绿叶突变体ygl11(t)的生理特性和基因克隆[J]. 中国水稻科学, 2015, 29(2): 111-118.

Shu-jun WU, Jie YANG, Ying YAN, Li-xia ZHANG, Fang-jun FAN, Jin-yan ZHU, Wen-qi LI, Wei-gong ZHONG, Li-ming CAO, Jun WANG. Physiological Characterization and Gene Identification of a Yellow-Green Leaf Mutant ygl11(t) in Rice[J]. Chinese Journal OF Rice Science, 2015, 29(2): 111-118.

图/表 9

图1 黄绿叶突变体ygl11(t)及野生型表型 A-分蘖期; B-灌浆期; C-成熟期叶片。

Fig. 1. Phenotype of the yellow-green leaf mutant and the wild type at different stages. A, Plant at tillering stage; B, Plant at grain filling stage; C, Leaf morphology at mature stages.

表1 黄绿叶突变体ygl11(t)及野生型的农艺性状

Table 1 Comparison of major agronomic traits between the ygl11(t)mutant and the wild type.

材料 Material	株高 Plant height /cm	有效穗数 Effective panicle number	穗长 Panicle length /cm	总粒数 Total grain number	结实率 Seed setting rate/%	千粒重 1000-grain weight/g
南粳41 Nanjing 41	93.3±0.5	10.3±0.3	17.4±1.2	162.0±9.5	89.5±2.1	27.7±0.3
ygl11(t)	108.1±0.7^**	9.2±0.2^**	21.5±0.9^**	105.1±6.3^**	87.0±2.3	26.8±0.3

表2 突变体ygl11(t)和南粳41叶片的叶绿素和类胡萝卜素含量

Table 2 Chlorophyll and carotenoid contents of ygl11(t) and the wild type at different growth stages.

时期 Stages	材料 Material	色素含量 Pigment content / (mg·g^-1)				叶绿素a/叶绿素b Chlorophyll a/ Chlorophyll b
时期 Stages	材料 Material	总叶绿素 Total chlorophyll	叶绿素a Chlorophyll a	叶绿素b Chlorophyll b	类胡萝卜素 Carotenoid	叶绿素a/叶绿素b Chlorophyll a/ Chlorophyll b
苗期 Seedling	NJ41	3.83±0.12	3.04±0.11	0.79±0.05	0.67±0.02	3.87±0.26
	ygl11(t)	2.86±0.11^**	2.66±0.10^**	0.20±0.01^**	0.65±0.02	13.02±0.45^**
分蘖盛期 Full tillering	NJ41	3.94±0.16	3.06±0.13	0.88±0.03	0.67±0.03	3.49±0.05
	ygl11(t)	1.80±0.02^**	1.69±0.02^**	0.11±0.00^**	0.42±0.01^**	14.99±0.52^**
齐穗期 Full heading	NJ41	3.84±0.03	3.01±0.02	0.83±0.01	0.69±0.01	3.60±0.04
	ygl11(t)	1.88±0.05^**	1.76±0.05^**	0.12±0.01^**	0.43±0.01^**	14.62±1.00^**

图2 ygl11(t)与南粳41不同生育时期的净光合速率 **表示在P=0.01 水平上差异显著(t测验)。

Fig. 2. Net photosynthetic rate of ygl11(t) and the wild type at different growth stages. **Significantly different at P=0.01(t test).

图3 ygl11(t)与野生型南粳41的叶绿体超微结构 A、B-南粳41的叶绿体结构;C、D-ygl11(t)的叶绿体结构。

Fig. 3. Ultrastructure of chloroplasts in the mesophyll cells of ygl11(t)and the wild type. A and B, Chloroplast structure of the wild type Nanjing 41; C and D, Chloroplast structure of the ygl11(t) mutant.

表3 ygl13(t)和9311正反交F2的表型分离

Table 3 Segregation of yellow-green leaf in F2 populations.

组合 Combination	总株数 Total number of plants	正常植株 No. of normal plants	黄绿植株 No. of yellow green plants	χ²(3:1)	$χ 0.05 2$
ygl11(t)/9311	1713	1305	408	1.21	3.84
9311/ygl11(t)	1243	942	301	0.37

表3 ygl13(t)和9311正反交F2的表型分离

Table 3 Segregation of yellow-green leaf in F2 populations.

组合 Combination	总株数 Total number of plants	正常植株 No. of normal plants	黄绿植株 No. of yellow green plants	χ²(3:1)	$χ 0.05 2$
ygl11(t)/9311	1713	1305	408	1.21	3.84
9311/ygl11(t)	1243	942	301	0.37

表4 YGL11(t)基因精细定位的引物

Table 4 The markers used for YGL11(t) gene mapping and sequencing.

引物名称 Primer name	类型 Type	正向引物(5'-3') Forward primer (5' - 3')	反向引物(5'-3') Reverse primer (5'-3')	酶切位点 Enzyme site
I1	InDel	CTCCGTCACTCTACTACCTGC	CTGCTGCGACATCTCACG	-
I4	InDel	ATATTGCAGGCCAACCATTC	CTCATTCTCATCCCTGTGCC	-
I6	InDel	GACGGACTTGAAGCGTAGCA	GCTGCATGTAGGACGACACC	-
I11	InDel	TTTAGGTGGAGCTGAAACTGT	TAAGCCAAACGATGGGAAC	-
D3	CAPs	CTCCCGAATCGAGTGGAATA	CAGATCGAGGGTGAAGAACC	BSHⅠ
D5	CAPs	ACAGCGTAACATTCAGCACC	CAACTTCTTGAGGAAAGGGTTA	SspⅠ
CAO1-1	CAO1测序	CGGCTGAGATGCGTTCC	GCACCGCATACACCTTGATT	-
CAO1-2	CAO1测序	CCCAGTAACCACAGGCTTATCT	ACTGAGCCAAGATGAAGAGGG	-
CAO1-3	CAO1测序	AAAGGATGGGAGACCTGGAT	TTCATCTCGCCAAGAAATACG	-
CAO1-4	CAO1测序	GGCAGCAAGAACGGATGAT	AGCACATAGATGGATAGAGGGAT	-
CAO2-1	CAO2测序	TATGGAACGCAGGAACGAG	TATGGAACGCAGGAACGAG	-
CAO2-2	CAO2测序	AGGTGGTAGAGGTTCTCAAATC	CATCAGTTGAATACTCCCACC	-
CAO2-3	CAO2测序	AAAGGCTCGGAACTGACATA	CGACAAGAGTTGAAAGAGGC	-
CAO2-4	CAO2测序	TGCCATTCAATAACCAAAGTG	CAAAGCCGACGCAGGTAG	-

图4 YGL11(t)基因的精细定位与克隆 A-YGL11(t)在第10染色体上初步定位结果; B-YGL11(t)的精细定位结果; C-YGL11(t)所在区域内的ORF; D-ygl11(t)与南粳41和日本晴的序列差异。

Fig. 4. Fine mapping and cloning of the YGL11(t) gene. A,Rough mapping of YGL11(t) on chromosome 10; B, Fine mapping of YGL11(t) gene; C,The ORFs in the 58.1 kb region; D, 2 bp deletion between ygl11(t)and Nanjing 41 and Nipponbare.

图5 ygl11(t)和日本晴OsCAO1基因的氨基酸序列比对结果

Fig. 5. Alignment of the OsCAO1amino acids in ygl11(t) and Nipponnbare in GenBank.

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