两个垩白突变体的鉴定及突变基因的图位克隆

doi:10.16819/j.1001-7216.2017.7003

摘要/Abstract

摘要：

【目的】研究两个水稻垩白突变体胚乳垩白的形成机制,为稻米品质改良提供理论基础。【方法】以从中花11的EMS突变体库中筛选出的两个稳定遗传的垩白突变体eb6、eb7为材料,对其进行农艺性状、稻米理化性质和遗传学分析,并利用eb6与南京11衍生的F₂群体对控制垩白的基因进行图位克隆。同时对候选基因的表达模式及淀粉合成相关基因在突变体及野生型中表达情况进行了分析。【结果】与野生型相比,两个突变体胚乳中央部位呈现白色且不透明,淀粉复合颗粒形状不规则且排列疏松,而突变体胚乳边缘部位与野生型无异,都为半透明状,淀粉复合颗粒呈多面体且排列致密。相对于野生型,突变体eb6、eb7成熟种子中的直链淀粉含量和胶稠度显著降低,蛋白质含量显著升高。RVA谱分析显示突变体淀粉黏滞性明显低于野生型。同时,支链淀粉聚合度分析显示突变体eb6中聚合度(DP)小于16的短链显著增加,DP为16~23的中长链显著减少。遗传分析表明突变体eb6、eb7胚乳垩白表型由单隐性核基因控制,并且它们为一对等位突变体。利用突变体eb6与南京11杂交衍生的F₂群体将突变体基因定位在第1染色体长臂上物理距离86.6 kb的区间内。该区间包含22个开放阅读框(ORF),其中ORF13编码腺苷二磷酸葡萄糖焦磷酸化酶大亚基2(OsAGPL2)。序列分析发现eb6与eb7分别在OsAGPL2第3、第7外显子上发生1个单碱基替换,并分别导致一个氨基酸替换。RT-PCR及原位杂交结果显示,OsAGPL2主要在水稻发育中的籽粒中表达。同时OsAGPL2的突变导致了多个淀粉合成相关基因在水稻籽粒灌浆过程中的表达模式发生改变。【结论】突变体eb6、eb7籽粒胚乳出现严重垩白表型为OsAGPL2突变所致。本研究进一步证明了OsAGPL2在调控水稻籽粒胚乳中淀粉的合成、淀粉复合颗粒的形成及稻米理化性质的平衡中起着重要的作用。

关键词: 垩白, 淀粉合成, 图位克隆, OsAGPL2, 水稻

Abstract:

【Objective】Chalkiness affects the appearance, processing, cooking and eating quality of rice. The objective of the study was to uncover the genetic mechanism of two rice chalky endosperm mutants for improving rice quality. 【Method】Two chalkiness mutants, eb6 and eb7 were identified from EMS-treated japonica rice Zhonghua 11. The agronomic traits and starch physicochemical properties of the two mutants were investigated. Genetic analysis and map-based cloning for the gene responsible for the eb6 and eb7 phenotypes were carried out with the F₂ population derived from the cross between eb6 and Nanjing 11, eb7 and 93-11. Furthermore, the expression pattern of candidate gene and the transcript levels of genes related to starch synthase in mutants and wild type(WT) were also investigated.【Result】The central parts of them were white and opaque, with loosely and irregularly arranged and smaller compound starch granules(SGs). Whereas, the marginal part of endosperms of WT and mutants and the central part of WT were filled with densely packed, similar sized polyhedral SGs. The amylose content and gel consistency of mutants were dramatically lower than those of WT. Simultaneously, the proportions of chains with degree of polymerization(DP) of amylopectin in the range from 6 to 16 were significantly increased, whereas the proportion of chains with DP in the range from 16 to 23 was noticeably decreased in the eb6. Genetic analysis showed that a single recessive gene controls chalkiness phenotype of mutants. Based on the F₂ population derived from the cross between eb6 and Nanjing 11, the gene was finally narrowed down to a 86.6 kb physical region on chromosome 1. Within this region, one open reading frame(LOC_Os01g44220) has been annotated as large subunit of ADP-glucose pyrophosphorylase gene(OsAGPL2). Sequence analysis revealed that there was only one a nucleotide substitution in the 3rd exon of eb6 and the 7th exon of eb7, which resulted in an amino acid replacement in OsAGPL2, respectively. The qRT-PCR and in-situ hybridization assay indicated that OsAGPL2 was mainly expressed in the developing grains. Moreover, the expression patterns of many genes involved starch synthesis were intensively influenced in mutants.【Conclusion】OsAGPL2 indisputably corresponds to endosperm chalkiness of eb6 and eb7. And all results suggest that OsAGPL2 plays an important role in starch synthesis, the formation of compound starch granules and rice quality.

Key words: chalkiness, starch synthesis, mapping-based cloning, OsAGPL2, rice

中图分类号:

张习春, 鲁菲菲, 吕育松, 罗荣剑, 焦桂爱, 邬亚文, 唐绍清, 胡培松, 魏祥进. 两个垩白突变体的鉴定及突变基因的图位克隆[J]. 中国水稻科学, 2017, 31(6): 568-579.

Xichun ZHANG, Feifei LU, Yusong LÜ, Rongjian LUO, Guiai JIAO, Yawen WU, Shaoqing TANG, Peisong HU, Xiangjin WEI. Identification and Gene Mapping-based Clone of Two Chalkiness Mutants in Rice[J]. Chinese Journal OF Rice Science, 2017, 31(6): 568-579.

图/表 10

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标记 Marker	正向引物序列 Forward(5′-3′)	反向引物序列 Reverse(5′-3′)	片段大小 Product size/bp
RM7405	TTGGCTCGCCCATATATAGG	CAGTCAGTCATCACTGGTAGTCG	92
RM7341	GCTTTGCTTGGTGGTCATTC	TGCAAGCTGAGTGTGAAACC	145
M1	TGCAACTTCTAGCTGCTCGA	GCATCCGATCTTGATGGG	112
M2	ACACCGCACCTAAACCCAAGACC	AGGCGCGTGAGAGAGGAATGG	151
M4	GACAACAATGGCCTCATCTTCC	GTCGTCGTCCTCGTAGTTCACG	99
M5	CCAGTGCACAACAGCACAAGC	GTTGGCTACTTGGCTTTCGATGG	151
M7	GTTCGTTTCCGAGATGTCACTGG	CGGGAGAGGGTACGATGAGTACG	157
M10	CTCCCCTTGTATTGGTAGA	TGAGAAACATCATACTCCATA	174
M11	CTCCCTCCCCAGGCAAGCAC	TGCAGCACAGACACGAGACC	106
M12	CATAACCCTGAAGTGGTGTGACG	GTGCTTGATGATATGGTCCTTGC	149
M13	GGAACTGCAGATTTGATGGAGAGG	CATATTCCACTGGGCGTCTGG	106
M16	AAAGCCTGGATAAGATGGTTCG	CTGTAGTTGCTGTTTGCCTGTCC	124
M17	GTGGAGGAGCGAAGGGAACACG	CCTCCCATATAAACCGGCGAACC	140
M19	ACGAGCACTACAGCACACATGC	AATGCTGCAACCTCTTCTTCTCC	144

标记 Marker	正向引物序列 Forward(5′-3′)	反向引物序列 Reverse(5′-3′)	片段大小 Product size/bp
RM7405	TTGGCTCGCCCATATATAGG	CAGTCAGTCATCACTGGTAGTCG	92
RM7341	GCTTTGCTTGGTGGTCATTC	TGCAAGCTGAGTGTGAAACC	145
M1	TGCAACTTCTAGCTGCTCGA	GCATCCGATCTTGATGGG	112
M2	ACACCGCACCTAAACCCAAGACC	AGGCGCGTGAGAGAGGAATGG	151
M4	GACAACAATGGCCTCATCTTCC	GTCGTCGTCCTCGTAGTTCACG	99
M5	CCAGTGCACAACAGCACAAGC	GTTGGCTACTTGGCTTTCGATGG	151
M7	GTTCGTTTCCGAGATGTCACTGG	CGGGAGAGGGTACGATGAGTACG	157
M10	CTCCCCTTGTATTGGTAGA	TGAGAAACATCATACTCCATA	174
M11	CTCCCTCCCCAGGCAAGCAC	TGCAGCACAGACACGAGACC	106
M12	CATAACCCTGAAGTGGTGTGACG	GTGCTTGATGATATGGTCCTTGC	149
M13	GGAACTGCAGATTTGATGGAGAGG	CATATTCCACTGGGCGTCTGG	106
M16	AAAGCCTGGATAAGATGGTTCG	CTGTAGTTGCTGTTTGCCTGTCC	124
M17	GTGGAGGAGCGAAGGGAACACG	CCTCCCATATAAACCGGCGAACC	140
M19	ACGAGCACTACAGCACACATGC	AATGCTGCAACCTCTTCTTCTCC	144

引物名称 Primer name		正向引物序列 Forward(5′-3′)	反向引物序列 Reverse(5′-3′)
Actin	X16280	CATGCTATCCCTCGTCTCGACCT	CGCACTTCATGATGGAGTTGTAT
OsAGPS1	AK073146	AGAATGCTCGTATTGGAGAAAATG	GGCAGCATGGAATAAACCAC
OsAGPS2a	AK071826	ACTCCAAGAGCTCGCAGACC	GCCTGTAGTTGGCACCCAGA
OsAGPS2b	AK103906	AACAATCGAAGCGCGAGAAA	GCCTGTAGTTGGCACCCAGA
OsAGPL1	AK069296	GGAAGACGGATGATCGAGAAAG	CACATGAGATGCACCAACGA
OsAGPL2	AK071497	GTTACTGGAACTGCACGATG	CTCCACCCAAAATGACAGC
OsAGPL3	AK100910	AAGCCAGCCATGACCATTTG	CACACGGTAGATTCACGAGACAA
OsAGPL4	AK121036	TCAACGTCGATGCAGCAAAT	ATCCCTCAGTTCCTAGCCTCATT
GBSSⅠ	AK070431	TCCGAGAGGTTCAGGTCATC	ATGAGCTCCTCGGCGTAGTA
OsSSI	AK109458	GGGCCTTCATGGATCAACC	CCGCTTCAAGCATCCTCATC
OsSSⅡa	AF419099	GCTTCCGGTTTGTGTGTTCA	CTTAATACTCCCTCAACTCCACCAT
OsISA1	AB093426	TGCTCAGCTACTCCTCCATCATC	AGGACCGCACAACTTCAACATA
OsISA2	AC132483	TAGAGGTCCTCTTGGAGG	AATCAGCTTCTGAGTCACCG
OsISA3	AP005574	ACAGCTTGAGACACTGGGTTGAG	GCATCAAGAGGACAACCATCTG
OsBEI	AK068920	CAAACTTTGACTAACAGGAGA	TTGATGGTAGGTGAAGCAG
OsBEⅡb	AK108535	ATGCTAGAGTTTGACCGC	AGTGTGATGGATCCTGCC
OsPHOL	AK063766	TTGGCAGGAAGGTTTCGCT	CGAAGCCTGAAGTGAACTTGCT

引物名称 Primer name		正向引物序列 Forward(5′-3′)	反向引物序列 Reverse(5′-3′)
Actin	X16280	CATGCTATCCCTCGTCTCGACCT	CGCACTTCATGATGGAGTTGTAT
OsAGPS1	AK073146	AGAATGCTCGTATTGGAGAAAATG	GGCAGCATGGAATAAACCAC
OsAGPS2a	AK071826	ACTCCAAGAGCTCGCAGACC	GCCTGTAGTTGGCACCCAGA
OsAGPS2b	AK103906	AACAATCGAAGCGCGAGAAA	GCCTGTAGTTGGCACCCAGA
OsAGPL1	AK069296	GGAAGACGGATGATCGAGAAAG	CACATGAGATGCACCAACGA
OsAGPL2	AK071497	GTTACTGGAACTGCACGATG	CTCCACCCAAAATGACAGC
OsAGPL3	AK100910	AAGCCAGCCATGACCATTTG	CACACGGTAGATTCACGAGACAA
OsAGPL4	AK121036	TCAACGTCGATGCAGCAAAT	ATCCCTCAGTTCCTAGCCTCATT
GBSSⅠ	AK070431	TCCGAGAGGTTCAGGTCATC	ATGAGCTCCTCGGCGTAGTA
OsSSI	AK109458	GGGCCTTCATGGATCAACC	CCGCTTCAAGCATCCTCATC
OsSSⅡa	AF419099	GCTTCCGGTTTGTGTGTTCA	CTTAATACTCCCTCAACTCCACCAT
OsISA1	AB093426	TGCTCAGCTACTCCTCCATCATC	AGGACCGCACAACTTCAACATA
OsISA2	AC132483	TAGAGGTCCTCTTGGAGG	AATCAGCTTCTGAGTCACCG
OsISA3	AP005574	ACAGCTTGAGACACTGGGTTGAG	GCATCAAGAGGACAACCATCTG
OsBEI	AK068920	CAAACTTTGACTAACAGGAGA	TTGATGGTAGGTGAAGCAG
OsBEⅡb	AK108535	ATGCTAGAGTTTGACCGC	AGTGTGATGGATCCTGCC
OsPHOL	AK063766	TTGGCAGGAAGGTTTCGCT	CGAAGCCTGAAGTGAACTTGCT

杂交组合 Cross	野生型植株 Number of wild type plants	突变体植株 Number of mutant plants	χ²_{(3:1 )}
eb6/南京11 eb6/Nanjing 11	310	119	1.72
eb7/93-11	253	96	1.17