籼稻93-11辐射诱变突变体库的创建及其筛选

doi:10.16819/j.1001-7216.2016.5126

摘要/Abstract

摘要：

人工诱变突变体库是植物种质资源创新和基因组功能分析的有力工具。本研究以具有优异农艺性状且已完成基因组测序的籼稻常规品种93-11(O. sativa ssp. indica cv. 93-11)为材料,创建了一个包含3617个家系的辐射诱变突变体库。通过在M₂和M₃代各生育期的筛选,从该突变体库中获得308个表型稳定的突变体。这些突变体的表型可以分成15类,包括株高、分蘖数、株型、穗型、小穗结构、育性、叶色、叶型、抽穗期、苯达松抗性等,突变频率为0.03%~2.74%。使用反向遗传学策略,鉴定出了2个雄性不育突变体和2个苯达松敏感突变体中的基因突变位点。其中,2个雄性不育突变体的突变表型是由于CYP704B2分别在第3和第4外显子发生碱基替换和片段缺失造成的,而2个苯达松敏感突变体的突变表型是由于CYP81A6中第1和第2外显子分别缺失了一个7 bp和一个23 bp的片段造成的。4个突变体家系中野生型和突变体植株的分离比均符合3:1,属单基因隐性突变。这一籼稻突变体库将有助于籼稻基因组的功能分析,并且为水稻遗传改良提供了有价值的材料。

关键词: 籼稻, 辐射诱变, 突变体库, 雄性不育, 苯达松敏感

Abstract:

Mutant libraries are powerful tools for plant germplasm creation and functional analysis of genome. Using 93-11 (O. sativa ssp. indica cv. 93-11), an elite variety with complete genome sequences available, we established an irradiation-induced mutant library containing 3617 lines. Three hundred and eight mutant lines were obtained from M₂ and M₃ generations and classfied into 15 types of mutation, including plant height, tiller number, plant architecture, inflorescence architecture, spikelet architecture, fertility, leaf color, leaf shape, heading date, and bentazon resistance. The mutation frequencies ranged from 0.03% to 2.74%. Using reverse genetics approach, we found that CYP704B2 is the causal gene for two male sterile mutants and CYP81A6 is responsible for two bentazon-lethal mutations. For the two male sterile mutants, nucleotide substitution and deletion were detected in the third and fourth exons of CYP704B2, respectively. For the two bentazon-lethal mutants, a 7-bp and a 23-bp deletion were detected in the first and second exons of CYP81A6, respectively. Genetic studies revealed that wild-type plants and mutant plants segregated at 3:1 ratio, suggesting that these mutant traits were controlled by single recessive genes. Our study showed that this mutant library is a useful resource for rice genome functional analysis and rice genetic improvement.

Key words: indica, mutagenesis by irradiation, mutant library, male sterility, bentazon-lethal

中图分类号:

龙湍, 安保光, 李新鹏, 张维, 李京琳, 杨瑶华, 曾翔, 吴永忠, 黄培劲. 籼稻93-11辐射诱变突变体库的创建及其筛选[J]. 中国水稻科学, 2016, 30(1): 44-52.

Tuan LONG, Bao-guang AN, Xin-peng LI, Wei ZHANG, Jing-lin LI, Yao-hua YANG, Xiang ZENG, Yong-zhong WU, Pei-jin HUANG. Construction and Screening of an Irradiation-induced Mutant Library of indica Rice 93-11[J]. Chinese Journal OF Rice Science, 2016, 30(1): 44-52.

图/表 5

表1 本研究使用的引物

Table 1 Primers used in this study.

引物 Primer	序列(5'-3') Sequence (5'-3')	目的基因 Target locus
704B2-1F	CAAAGATTGTCTCAAGGTTGGTAG	CYP704B2
704B2-1R	GGTATTAGGCAAGGAATTCAGTTG
704B2-2F	TCGAAGGACAGGACGGTGAC	CYP704B2
704B2-2R	TTTGAGCAAGAGAGGAAGGATC
704B2-3F	GCAAGAACTAACCAAAATTCAGG	CYP704B2
704B2-3R	GGTCAGACGGAGGTGGAGA
81A6-F1	CAAACTTCCAACTTTCCCGT	CYP81A6
81A6-R1	CTGACGCCTGAAACGCAG
81A6-F2	GCAACGGAGTGAGTAGAAGTAATC	CYP81A6
81A6-R2	CAGAAAGGTAAAACAGCAGAAGA
81A6-F3	GGGCGAGAAGAAGAGCATG	CYP81A6
81A6-R3	GGCACGACCTTGGGGAT
81A6-F4	GGTGCGACGGCAATCTC	CYP81A6
81A6-R4	GAGCAGAAAGCCCTTCCTC
1907_F	AGGTCGGGTTTGGGGTT	1907株系中CYP704B2的突变位点
1907_R	GATGTTGGCAGCGTCGAA	CYP704B2 in the line 1907
2245_F	AGCTTCGGGGACGACAAGA	2245株系中CYP704B2的突变位点
2245_R	TGCGTCATCGCCATGTACGT	CYP704B2 in the line 2245
3522-F	ATGAAATCTTAGTTCCACCCTCTTGCCG	3522株系中CYP81A6的突变位点
3522-R	CTCCCTGACGATGCACTGGAGGT	CYP81A6 in the line 3522
3622-F	TGTTCGAGGTCTCCCTCAGCGTG	3622株系中CYP81A6的突变位点
3622-R	CCTGCTTAAACTCCTGGGCTTCCA	CYP81A6 in the line 3622

图1 M2家系中不同类型的突变体 A-单分蘖; B-矮化; C-迟抽穗; D-小穗变形; E-假病斑。左边为突变体,右边为野生型。

Fig. 1. Different mutant phenotypes in M2 families. A, Monoculm; B, Dwarfism; C, Late heading; D, Malformed spikelet; E, Lesion mimic. Mutant plants or samples are on the left, and wild type plants or samples are on the right.

表2 M2和M3家系中突变表型统计

Table 2 Summery of mutant phenotypes in M2 and M3 families.

表型 Phenotype	家系数 Number of lines	频率^a Frequency ^a /%
株高增加Increased plant height	3	0.08
矮化 Dwarfism	65	1.80
株型紧凑 Reduced tiller angle	1	0.03
分蘖少 Reduced tiller number	52	1.44
分蘖多 Increased tiller number	2	0.06
穗型变异 Malformed panicle	6	0.17
小穗变异 Malformed spikelet	19	0.53
育性 Fertility	99	2.74
叶色 Leaf color	2	0.06
叶片变宽 Broad leaf	2	0.06
假病斑 Lesion mimic	12	0.33
早衰 Premature senescence	1	0.03
早抽穗 Early heading	12	0.33
迟抽穗 Late heading	30	0.83
苯达松敏感 Bentazon-lethal	2	0.15^b
总数 Total	308	8.52

图2 无花粉型雄性不育突变体及其分子鉴定 A和B-1907家系中野生型(A)和无花粉型不育突变体(B)的花粉碘染,比例尺=100 μm;C和D-2245家系中野生型(C)和无花粉型不育突变体(D)的花粉碘染,比例尺=100 μm;E-CYP704B2的基因结构。黑框代表编码区;线条代表内含子;三角指向突变位点,其中1907家系突变体(1907m)中CYP704B2第794个碱基之后的“GGG”被替换为“T”,而2245家系突变体(2245m)中CYP704B2第1267个碱基之后的“GG”缺失;F和G-1907(F)和2245(G)家系M2单株中CYP704B2突变位点的基因型检测。图片左边显示PCR产物大小。W-野生型;H-杂合型;M-突变型。

Fig. 2. Phenotypic and molecular characterization of two male-sterile mutants without pollen grains. A and B, I2-KI staining of pollen grains of a wild type plant (A) and a mutant (B) in the line 1907, bar = 100 μm; C and D, I2-KI staining of pollen grains of a wild type plant (C) and a mutant (D) in the line 2245, bar = 100 μm; E, The structure of CYP704B2. Black boxes represent the coding region; Lines represent introns; Triangles point to the mutation loci. “GGG” following the 794th nucleotide of CYP704B2 is substituted by “T” in mutants of the line 1907 (1907m), and “GG” following the 1267th nucleotide of CYP704B2 is deleted in mutants of the line 2245 (2245m); F and G, Genotyping of the CYP704B2 loci in the lines 1907 (F) and 2245 (G). The sizes (bp) of the PCR products are shown on the left. W, Wild type; H, Heterozygote; M, Mutant.

图3 苯达松敏感突变体及其分子鉴定 A和B-3522(A)和3622(B)家系中苯达松敏感突变体(左边)和野生型(右边)植株分蘖期喷施苯达松后的表型; C-CYP81A6的基因结构。黑框代表编码区;线条代表内含子;三角指向突变位点,其中3522家系突变体(3522m)中CYP81A6编码区第1747 bp后发生了23 bp缺失,而3622家系突变体(3622m)中CYP81A6编码区第586 bp后发生了7 bp缺失;D和E-3522(D)和3622(E)家系M3单株中CYP81A6突变位点的基因型检测。图片左边显示PCR产物大小。W-野生型;H-杂合型;M-突变型。

Fig. 3. Phenotypic and molecular characterization of two bentazon-lethal mutants. A and B, Phenotypes of the bentazon-lethal mutants (left) and wild type (right) plants in the lines 3522 (A) and 3622 (B) after bentazon spraying at tillering stage; C, The structure of CYP81A6. Black boxes represent the coding region; Lines represent introns; Triangles point to the mutation loci. A 23-bp deletion is found following the 1747th bp of the coding region of CYP81A6 in mutants of the line 3522 (3522m), and a 7-bp deletion is found following the 586th bp of the coding region of CYP81A6 in mutants of the line 3622 (3622m); D and E, Genotyping of the CYP81A6 loci in the lines 3522 (D) and 3622 (E). The sizes (bp) of the PCR products are shown on the left. W, Wild type; H, Heterozygote; M, Mutant.

参考文献 27

[1]	Zhang Q, Li J, Xue Y, et al.Rice 2020: A call for an international coordinated effort in rice functional genomics.Mol Plant, 2008, 1(5): 715-719.
[2]	Goff S A, Ricke D, Lan T H, et al.A draft sequence of the rice genome (Oryza sativa L. ssp. japonica).Science, 2002, 296(5565): 92-100.
[3]	Yu J, Hu S, Wang J, et al.A draft sequence of the rice genome (Oryza sativa L. ssp. indica).Science, 2002, 296(5565): 79-92.
[4]	Kawahara Y, de la Bastide M, Hamilton J P, et al. Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data.Rice, 2013, 6(1): 4.
[5]	IRGSP. The map-based sequence of the rice genome.Nature, 2005, 436(7052): 793-800.
[6]	Wang N, Long T, Yao W, et al.Mutant resources for the functional analysis of the rice genome.Mol Plant, 2013, 6(3): 596-604.
[7]	Jeong D H, An S, Kang H G, et al.T-DNA insertional mutagenesis for activation tagging in rice.Plant Physiol, 2002, 130: 1636-1644.
[8]	Miyao A, Tanaka K, Murata K, et al.Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome.Plant Cell, 2003, 15: 1771-1780.
[9]	Wu C, Li X, Yuan W, et al.Development of enhancer trap lines for functional analysis of the rice genome.Plant J, 2003, 35(3): 418-427.
[10]	Kolesnik T, Szeverenyi I, Bachmann D, et al.Establishing an efficient Ac/Ds tagging system in rice: Large-scale analysis of Ds flanking sequences.Plant J, 2004, 37(2): 301-314.
[11]	Sallaud C, Gay C, Larmande P, et al.High throughput T-DNA insertion mutagenesis in rice: A first step towards in silico reverse genetics.Plant J, 2004, 39: 450-464.
[12]	Hsing Y I, Chern C G, Fan M J, et al.A rice gene activation/knockout mutant resource for high throughput functional genomics.Plant Mol Biol, 2007, 63: 351-364.
[13]	Fu F F, Ye R, Xu S P, et al.Studies on rice seed quality through analysis of a large-scale T-DNA insertion population.Cell Res, 2009, 19(3): 380-391.
[14]	Wei F J, Droc G, Guiderdoni E, et al.International consortium of rice mutagenesis: Resources and beyond.Rice, 2013, 6(1): 39.
[15]	Till B J, Cooper J, Tai T H, et al.Discovery of chemically induced mutations in rice by TILLING.BMC Plant Biol, 2007, 7: 19-30.
[16]	Suzuki T, Eiguchi M, Kumamaru T, et al.MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice.Mol Genet Genom, 2008, 279(3): 213-223.
[17]	Abe A, Kosugi S, Yoshida K, et al.Genome sequencing reveals agronomically important loci in rice using MutMap.Nat Biotechnol, 2012, 30(2): 174-178.
[18]	Fekih R, Takagi H, Tamiru M, et al.MutMap+: Genetic mapping and mutant identification without crossing in rice.PLoS One, 2013, 8(7): e68529.
[19]	Wu J L, Wu C, Lei C, et al.Chemical- and irradiation-induced mutants of indica rice IR64 for forward and reverse genetics.Plant Mol Biol, 2005, 59(1): 85-97.
[20]	Rogers S O, Bendich A J.Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues.Plant Mol Biol, 1985, 5(2): 69-76.
[21]	Li H, Pinot F, Sauveplane V, et al.Cytochrome P450 family member CYP704B2 catalyzes the ω-hydroxylation of fatty acids and is required for anther cutin biosynthesis and pollen exine formation in rice.Plant Cell, 2010, 22(1): 173-190.
[22]	张集文. 水稻苯达松敏感突变研究进展. 中国水稻科学, 2010, 24(5): 551-558.
	Zhang J W.Progress on the study of the bentazon sensitive mutants in rice.Chin J Rice Sci, 2010, 24(5): 551-558. (in Chinese with English abstract)
[23]	Liu L, Fan X D.Tapetum: Regulation and role in sporopollenin biosynthesis in Arabidopsis.Plant Mol Biol, 2013, 83(3): 165-175.
[24]	马西青, 方才臣, 邓联武, 等. 水稻隐性核雄性不育基因研究进展及育种应用探讨. 中国水稻科学, 2012, 26(5): 511-520.
	Ma X Q, Fang C C, Deng L W, et al.Research progress and breeding application of recessive genic male sterility genes in rice.Chin J Rice Sci, 2012, 26(5): 511-520. (in Chinese with English abstract)
[25]	邓兴旺, 王海洋, 唐晓艳, 等. 杂交水稻育种将迎来新时代. 中国科学:生命科学, 2013, 43(10): 864-868.
	Deng X W, Wang H Y, Tang X Y, et al.Hybrid rice breeding welcomes a new era of molecular crop design.Sci Sin Vitae, 2013, 43(10): 864-868. (in Chinese with English abstract)
[26]	汪旭东, 周开达, 李仕贵, 等. 利用隐性核不育性进行水稻轮回育种初步研究. 西南农业学报, 2001, 14(3): 102-106.
	Wang X D, Zhou K D, Li S G, et al.A preliminary study on rice recurrent breeding using recessive sterile material.Southwest Chin J Agri Sci, 2001, 14(3): 102-106. (in Chinese with English abstract)
[27]	Wu Y, Fox T W, Trimnell M R, et al.Development of a novel recessive genetic male sterility system for hybrid seed production in maize and other cross-pollinating crops.Plant Biotechnol J, 2015, doi: 10.1111/pbi.12477.