CN108588086B - 水稻mapk6基因突变体及其应用 - Google Patents
水稻mapk6基因突变体及其应用 Download PDFInfo
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- CN108588086B CN108588086B CN201810367176.9A CN201810367176A CN108588086B CN 108588086 B CN108588086 B CN 108588086B CN 201810367176 A CN201810367176 A CN 201810367176A CN 108588086 B CN108588086 B CN 108588086B
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Abstract
本发明提供水稻MAPK6基因突变体及其应用,属于植物基因工程技术领域。本发明的水稻MAPK6基因突变体分别为DN‑OsMAPK6和CA‑OsMAPK6,其核苷酸序列如SEQ ID NO.2、4所示,本发明证实突变体DN‑OsMAPK6转入水稻后具有使水稻籽粒变小的功能,突变体CA‑OsMAPK6转入水稻后具有使水稻籽粒变大的功能,在水稻种质资源的遗传改良育种中作用重大。本发明还提供了该突变体在水稻育种制种中的应用。
Description
技术领域
本发明属于植物基因工程领域,具体地说,涉及水稻MAPK6基因突变体MAPK6及其在影响水稻籽粒大小中的应用。
背景技术
水稻(Oryza sativa L.)是我国和全世界最重要的三大粮食作物之一,是世界一半以上人口的主食,也是一个重要的功能基因研究的模式植物。随着耕地面积的减少和人口的快速增长,提高水稻产量对保证我国粮食安全具有十分重要的意义。当前水稻增产的研究较依赖于有限的水稻种质资源,传统的杂交育种优势正在逐渐减弱,而水稻转基因技术有可能发掘水稻进一步增产的潜力。
在植物界中,能形成种子的植物约占植物总数的三分之二以上,作为重要的繁殖器官,种子同时也为人们提供食物来源,水稻就是其中的重要代表。水稻产量是由有效分蘖数、穗粒数和粒型所决定。水稻粒型包括籽粒的长度、宽度和厚度。通过增加水稻籽粒的长度和宽度可以有效的提高水稻产量。水稻中已经发现了一些与粒型相关的基因,例如:GS3,GW2,GW5,GS5,GW8,GL7等。所以,研究植物体如何调控器官大小的机制已经成为提高作物产量的重要策略之一。MAPK信号通路是高度保守的信号通路,参与植物生长发育的多个过程。研究显示OsMAPK6部分丢失功能会导致水稻的籽粒变小(OsMAPK6,a mitogen-activatedprotein kinase,influences rice grain size and biomass production.PlantJ.2015,84(4):672-81.doi:10.1111/tpj.13025)。然而本领域技术人员仍然无从知悉关于OsMAPK6基因的哪些突变能够影响籽粒变大或变小。若能够通过定点突变实现OsMAPK6功能的丢失或加强,则有助于水稻的改良育种和制种工作。
发明内容
本发明目的是提供水稻MAPK6基因突变体及其应用。
本发明提供了两个水稻MAPK6基因突变体,其中突变体DN-OsMAPK6为水稻MAPK6基因(其CDS序列如SEQ ID NO.5所示)第673位处的A突变为G,和第680位的A突变为T。
水稻MAPK6基因突变体CA-OsMAPK6为水稻MAPK6基因第443位处的A突变为G。
进一步地,本发明提供的水稻MAPK6基因突变体,其为DN-OsMAPK6,其氨基酸序列如SEQ ID NO.1所示或如SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的具有与所述DN-OsMAPK6突变体相同功能的氨基酸序列。
更进一步地,水稻MAPK6基因突变体DN-OsMAPK6的核苷酸序列如SEQ ID NO.2所示。
本发明提供的另一个水稻MAPK6基因突变体CA-OsMAPK6,其氨基酸序列如SEQ IDNO.3所示或如SEQ ID NO.3所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的具有与所述CA-OsMAPK6突变体相同功能的氨基酸序列。
进一步地,水稻MAPK6基因突变体的核苷酸序列如SEQ ID NO.4所示。
含有上述水稻MAPK6基因突变体的表达载体属于本发明的保护范围。
进一步地,含有所述表达载体的宿主细胞属于本发明的保护范围。
本发明还提供了水稻MAPK6基因突变体DN-OsMAPK6在使植物籽粒长度、宽度、厚度或/和重量减小中的应用。
本发明提供了水稻MAPK6基因突变体CA-OsMAPK6在使植物籽粒长度、宽度、厚度或/和重量增加中的应用。
本发明提供了上述两种水稻MAPK6基因突变体在制备转基因植物中的应用。
优选地,所述转基因植物为转基因水稻。
更优选地,本发明提供了水稻MAPK6基因突变体CA-OsMAPK6在制备籽粒更大的转基因水稻中的应用。
本发明提供了水稻MAPK6基因突变体DN-OsMAPK6在制备籽粒更小的转基因植物中的应用。
本发明还提供了上述两种水稻MAPK6基因突变体在农作物改良育种、制种中的应用。
优选地,所述的农作物为水稻、玉米、小麦、棉花。
本发明的上述两种水稻MAPK6基因突变体在水稻种质资源改良方面的用途属于本发明的保护范围。
本发明的优点在于:(1)本发明的水稻MAPK6基因突变体DN-OsMAPK6和CA-OsMAPK6来源于水稻,对水稻分子育种十分有利。(2)突变体与野生型相比仅有1个碱基的突变,采用测序即能够实现鉴别,不需要特别的检测技术和方法。(3)本发明的两个水稻MAPK6基因突变体转入水稻后,能够使水稻相比野生型的籽粒的形状发生明显变大或变小,籽粒变大有利于水稻产量的提高;(4)本发明的水稻MAPK6基因突变体在水稻种质资源改良中效果明显,经济价值巨大。
附图说明
图1为转基因植株的表型鉴定,其中A到C是转DN-OsMAPK6基因植株的籽粒变小:A图中的左侧两个是作为转基因受体的水稻品种中花11(ZH11)的籽粒,右侧两个为T0代转DN-OsMAPK6基因植株的籽粒;B为中花11和T0代转DN-OsMAPK6基因植株的籽粒的大小测量比较;C是中花11和T0代转DN-OsMAPK6基因植株的OsMAPK6和DN-OsMAPK6的总表达表达量检测结果;B和C中每一组柱中左侧柱为中花11的籽粒,右侧柱为DN-OsMAPK6基因植株的籽粒。D到F反应的是转CA-OsMAPK6基因植株的籽粒变大:D的左侧两个为转基因受体的水稻品种中花11(ZH11)的籽粒,右侧两个为T0代转CA-OsMAPK6基因植株的籽粒;E代表中花11和T0代转CA-OsMAPK6基因植株的籽粒的大小测量比较;F是中花11和T0代转CA-OsMAPK6基因植株的OsMAPK6和DN-OsMAPK6的总表达表达量检测结果;E和F中每一组柱中左侧柱为中花11的籽粒,右侧柱为CA-OsMAPK6基因植株的籽粒。**代表差异显著。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段;若未特别指明,实施例中所用试剂均为市售。
实施例中的pIKPB003(A set of modular binary vectors for transformationof cereals.Plant Physiol.145(4),1192-1200(2007)),实施例中水稻品种中花11(朱旭东,陈红旗,罗达,张建军,方红民,闵绍楷。水稻中花11辐射突变体的分离与鉴定。中国水稻科学,2003,17(3):205-210),实施例中根癌农杆菌GV3101(Li,Y.,Zheng,L.,Corke,F.,Smith,C.,and Bevan,M.W.(2008)Control of final seed and organ size by theDA1gene family in Arabidopsis thaliana.Genes Dev22,1331-1336)公众可从中国科学院遗传与发育生物学研究所获得,该生物材料只为重复本发明的相关实验所用,不可作为其它用途使用。
实施例1 DN-OsMAPK6编码基因和CA-OsMAPK6编码基因的获得
1、提取RNA
液氮研碎水稻中花11的叶片,用天根的植物总RNA提取试剂盒(TIANGEN),提取总RNA。将得到的总RNA用分光光度计(Eppendorf公司,德国)检测样品中总RNA的浓度。
2、DN-OsMAPK6编码基因的获得
根据RAP-DB(http://rapdb.dna.affrc.go.jp/)上的OsMAPK6基因序列设计两对引物如下:
OsMAPK6-F1:GGCTGCAGGAATTCAAGCTTATGGACGCCGGGGCGCAGC
OsMAPK6-R2:CAGATCAGTATCCATCAATTCACATGCAATATAAACGTCATTGA
DN-OsMAPK6-F:GCTGAGTTTGTTGTCACAAGATGGTAT
DN-OsMAPK6-R:CCATCTTGTGACAACAAACTCAGCCATAAAATCGGTTTCTGAGGT
取5μg总RNA,用反转录试剂盒(Invitrogen)进行反转录,以反转录得到的cDNA为模板,以OsMAPK6-F1和DN-OsMAPK6-R扩增得到片段A,以DN-OsMAPK6-F和OsMAPK6-R2扩增得到片段B。用琼脂糖凝胶回收试剂盒(TIANGEN)分别回收A片段和B片段。
3、CA-OsMAPK6基因的获得
根据RAP-DB(http://rapdb.dna.affrc.go.jp/)上的OsMAPK6基因序列设计两对引物如下:
OsMAPK6-F1:GGCTGCAGGAATTCAAGCTTATGGACGCCGGGGCGCAGC
OsMAPK6-R2:CAGATCAGTATCCATCAATTCACATGCAATATAAACGTCATTGA
CA-OsMAPK6-R1:ATCCAGTCACTATGGTCGACCTACTGGTAATCAGGGTTGAA
CA-OsMAPK6-F2:GAATTGATGGATACTGATCTG
以步骤2获得的cDNA为模板,以OsMAPK6-F1和CA-OsMAPK6-R1扩增得到片段C,以CA-OsMAPK6-F2和OsMAPK6-R2扩增得到片段D。用琼脂糖凝胶回收试剂盒(TIANGEN)分别回收C片段和D片段。
4、OsMAPK6基因的获得
以步骤2获得的cDNA为模板,以OsMAPK6-F1和OsMAPK6-R2为引物扩增得到OsMAPK6基因。
实施例2 DN-OsMAPK6和CA-OsMAPK6表达载体的构建
用无缝克隆试剂盒(TIANGEN)将实施例1回收的A片段和B片段同时与pIPKB003做无缝克隆连接,将连接产物转化大肠杆菌DH5α感受态细胞,根据载体上的壮观霉素抗性标记筛选阳性克隆,得到含有A和B片段融合的重组质粒,命名为pIPKB003-DN-OsMAPK6。
用无缝克隆试剂盒(TIANGEN)将实施例1回收的片段C和D同时与pIPKB003做无缝克隆连接,将连接产物转化大肠杆菌DH5α感受态细胞,根据载体上的壮观霉素抗性标记筛选阳性克隆,得到含有C和D片段融合的重组质粒,命名为pIPKB003-CA-OsMAPK6。
分别将pIPKB003-DN-OsMAPK6和pIPKB003-CA-OsMAPK6导入根癌农杆菌GV3101,分别得到含有pIPKB003-DN-OsMAPK6和pIPKB003-CA-OsMAPK6的重组根癌农杆菌菌株,将得到的菌株分别命名为GV3101-DN-OsMAPK6和GV3101-CA-OsMAPK6。
GV3101-OsMAPK6的构建方法同GV3101-DN-OsMAPK6和GV3101-CA-OsMAPK6。
实施例3转基因植株的获得
用GV3101-DN-OsMAPK6和GV3101-CA-OsMAPK6分别转化水稻品种中花11,以分别获得转DN-OsMAPK6和CA-OsMAPK6的转基因植株。具体操作如下:
1、水稻成熟胚愈伤组织的诱导培养:去壳的水稻成熟种子先用70%乙醇浸泡1-2min,然后用0.1%升汞浸泡10min,进行表面灭菌,无菌水冲洗3-4次,再将种子放在无菌滤纸上吸干水分后,放在成熟胚愈伤诱导培养基上,26℃暗培养。约10-15天后,剥下成熟胚盾片长出的愈伤组织,转入成熟胚继代培养基上,在相同条件下继代培养。以后每两周继代培养一次。挑选继代培养5-7天、色泽淡黄的愈伤组织共培养。
2、农杆菌的培养
将GV3101-DN-OsMAPK6和GV3101-CA-OsMAPK6在含有50mg/L壮观霉素的LB平板上划线,28℃黑暗培养3天,用一金属匙收集农杆菌菌体,将其悬浮于共培养CM液体培养基中,调整菌体浓度至OD600为0.3-0.5,加入乙酰丁香酮,使乙酰丁香酮终浓度为100mΜ,即为共培养转化水稻用的农杆菌悬浮液。
3、水稻愈伤组织与农杆菌的共培养
挑选状态较好(继代培养5-7天、色泽淡黄)的愈伤组织放入100ml无菌三角瓶中,加入适量农杆菌悬浮液(保证有足够的菌液与材料接触即可),室温放置20min,并不时晃动。倒掉菌液,将愈伤组织放在无菌滤纸上吸去多余菌液,随即转移到铺有一层无菌滤纸的固体共培养基上,26℃黑暗培养2-3天。
4、抗性愈伤组织的筛选
将共培养后的愈伤组织放在含有50mg/l潮霉素的筛选培养基上,26℃暗培养14天,转到新鲜配制的筛选培养基上继续筛选14天。大部分愈伤组织在筛选后10天左右褐化,然后在褐化组织的边缘重新生长出乳白色的抗性愈伤组织。
5、抗性愈伤组织的分化
从经两轮筛选后长出的抗性愈伤组织中,挑选乳黄色致密的抗性愈伤组织转至含有50mg/L潮霉素的分化培养基上,先暗培养3天,然后转至15h/d光照条件下培养,一般经过15-25天左右,有绿点出现。30-40天后进一步分化出小苗。
6、生根、壮苗和移栽
当抗性愈伤组织分化的芽长至约2cm时,将小苗移到生根培养基上,培养两周左右。选择高约10cm、根系发达的小苗,洗去培养基,移栽至田间。分别得到30株T0代转DN-OsMAPK6和CA-OsMAPK6基因的植株。
其中,所用的培养基配方如下:
诱导培养基:N6大量元素+MS-Fe盐+B5微量元素+B5有机+2,4-D 2.5mg/L+proline500mg/l+glutamine 500mg/l+CH 300mg/l+麦芽糖/蔗糖30g/l+Gelrite 2.6mg/l,pH 5.8。
继代培养基:同诱导培养基,但是2,4-D改为2.0mg/L。
共培养(固体)培养基:N6大量元素+MS-Fe盐+B5微量元素+B5有机+2,4-D 2.0mg/L+CH 500mg/l+肌醇2000mg/L+AS100μM+麦芽糖/蔗糖30g/l+Gelrite 2.6mg/l,pH 5.5。(液体选择培养基无Gelrite)。
筛选培养基:N6大量元素+MS-Fe盐+B5微量元素+B5有机+2,4-D 2.0mg/L+proline500mg/l+glutamine 500mg/l+CH 300mg/l+麦芽糖/蔗糖30g/l+Gelrite 2.6mg/l+cef250mg/l+Hyg 50mg/l,pH 5.8。
分化培养基:N6大量元素+MS-Fe盐+B5微量元素+B5有机+NAA 0.1mg/L+KT 4mg/L+proline 500mg/l+glutamine 500mg/l+CH 300mg/l+麦芽糖/蔗糖30g/l+Gelrite 2.6mg/l+cef.250mg/l+Hyg 50mg/l,pH 5.8。
生根培养基:1/2N6大量元素+MS-Fe盐+B5微量元素+蔗糖30g/l+Agar 0.8%,pH5.8。
7、转DN-OsMAPK6基因和转CA-OsMAPK6基因植株的鉴定
分别将步骤6得到的30株T0代转DN-OsMAPK6基因和转CA-OsMAPK6基因的植株通过PCR来进一步鉴定。以新鲜的转基因植株叶片的基因组DNA为模板,用潮霉素引物对HYG-F/HYG-R进行PCR扩增。转基因植株可以扩增出324bp条带。
HYG-F:GTCCATCACAGTTTGCCAGT
HYG-R:AGATCGTTATGTTTATCGGCACT
8、表达量分析:提取转基因植株和对照中花11叶片RNA,反转录后通过real-timePCR(Lightcycler 480,ROCHE)来分析转基因植株表达量。荧光染料为Lightcycler480SYBR GreenIMaster(ROCHE)。内参为ACTIN1。real-time PCR引物如下:
OsMAPK6RT-F:AGACTTGAAGCCCAGCAACCT
OsMAPK6RT-R:AGTTCCGGTGCCCTATACCAT
ACTIN1F:TGCTATGTACGTCGCCATCCAG
ACTIN1R:AATGAGTAACCACGCTCCGTCA
转OsMAPK6基因植株的获得同转DN-OsMAPK6和CA-OsMAPK6。
实施例4转基因植株的表型鉴定
将实施例3的得到的T0代转基因植株和作为转基因受体的水稻品种中花11在田间自然条件下生长,成熟后收取转基因植株和野生型对照中花11的籽粒,放在体式镜(LEICAS8APO,德国)下观察并拍照(LEICA DFC420,德国)。用Image J1.41软件测量籽粒的长度,利用EXCEL进行统计分析。
结果表明,与野生型对照相比,转DN-OsMAPK6基因植株的籽粒显著变小。转CA-OsMAPK6基因植株的籽粒显著变大,而转野生型OsMAPK6仅能够略微增加籽粒的长度(约提高3%左右)。结果如图1和表1所示。
表1转OsMAPK6基因植物的表型统计(**代表与野生型相比有显著差异)
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国科学院遗传与发育生物学研究所
<120> 水稻MAPK6基因突变体及其应用
<130> KHP181112207.3
<160> 19
<170> SIPOSequenceListing 1.0
<210> 1
<211> 398
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Asp Ala Gly Ala Gln Pro Pro Asp Thr Glu Met Ala Glu Ala Gly
1 5 10 15
Gly Gly Gln Gln Pro Pro Ala Ala Ala Ala Ala Ala Gly Ala Gly Ala
20 25 30
Gly Ala Gly Met Met Glu Asn Ile Gln Ala Thr Leu Ser His Gly Gly
35 40 45
Arg Phe Ile Gln Tyr Asn Ile Phe Gly Asn Val Phe Glu Val Thr Ala
50 55 60
Lys Tyr Lys Pro Pro Ile Leu Pro Ile Gly Lys Gly Ala Tyr Gly Ile
65 70 75 80
Val Cys Ser Ala Leu Asn Ser Glu Thr Gly Glu Gln Val Ala Ile Lys
85 90 95
Lys Ile Ala Asn Ala Phe Asp Asn Lys Ile Asp Ala Lys Arg Thr Leu
100 105 110
Arg Glu Ile Lys Leu Leu Arg His Met Asp His Glu Asn Ile Val Ala
115 120 125
Ile Arg Asp Ile Ile Pro Pro Pro Gln Arg Asn Ser Phe Asn Asp Val
130 135 140
Tyr Ile Ala Tyr Glu Leu Met Asp Thr Asp Leu His Gln Ile Ile Arg
145 150 155 160
Ser Asn Gln Ala Leu Ser Glu Glu His Cys Gln Tyr Phe Leu Tyr Gln
165 170 175
Ile Leu Arg Gly Leu Lys Tyr Ile His Ser Ala Asn Val Leu His Arg
180 185 190
Asp Leu Lys Pro Ser Asn Leu Leu Leu Asn Ala Asn Cys Asp Leu Lys
195 200 205
Ile Cys Asp Phe Gly Leu Ala Arg Thr Thr Ser Glu Thr Asp Phe Met
210 215 220
Ala Glu Phe Val Val Thr Arg Trp Tyr Arg Ala Pro Glu Leu Leu Leu
225 230 235 240
Asn Ser Ser Glu Tyr Thr Ala Ala Ile Asp Val Trp Ser Val Gly Cys
245 250 255
Ile Phe Met Glu Leu Met Asp Arg Lys Pro Leu Phe Pro Gly Arg Asp
260 265 270
His Val His Gln Leu Arg Leu Leu Met Glu Leu Ile Gly Thr Pro Asn
275 280 285
Glu Ala Asp Leu Asp Phe Val Asn Glu Asn Ala Arg Arg Tyr Ile Arg
290 295 300
Gln Leu Pro Arg His Ala Arg Gln Ser Phe Pro Glu Lys Phe Pro His
305 310 315 320
Val His Pro Leu Ala Ile Asp Leu Val Glu Lys Met Leu Thr Phe Asp
325 330 335
Pro Arg Gln Arg Ile Thr Val Glu Gly Ala Leu Ala His Pro Tyr Leu
340 345 350
Ala Ser Leu His Asp Ile Ser Asp Glu Pro Val Cys Ser Ser Pro Phe
355 360 365
Ser Phe Asp Phe Glu Gln His Ala Leu Ser Glu Glu Gln Met Lys Asp
370 375 380
Leu Ile Tyr Gln Glu Gly Leu Ala Phe Asn Pro Asp Tyr Gln
385 390 395
<210> 2
<211> 1197
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggacgccg gggcgcagcc gccggacacg gagatggcgg aggccggcgg cgggcagcag 60
ccgcctgctg cggctgcggc ggcgggggcg ggggcagggg cggggatgat ggagaacatc 120
caggcgacgc tgagccatgg cgggaggttc atccagtaca acatcttcgg gaacgtgttc 180
gaggtcaccg ccaagtacaa gccccccatc ctccccatcg gcaagggcgc ctacggcatc 240
gtctgctcgg cgctcaactc ggagacgggg gagcaggtgg cgatcaagaa gatcgccaac 300
gcgttcgaca acaagatcga cgccaagcgc acgctcaggg agatcaagct gctccgccac 360
atggaccacg agaatattgt tgccataagg gatatcatac ctcctccaca aaggaattca 420
ttcaatgacg tttatattgc atatgaattg atggatactg atctgcatca aattattcgc 480
tcaaatcaag cattgtcaga ggagcactgc cagtatttcc tttatcagat tctccgtggc 540
ttgaagtata tacattcagc aaatgtcctt caccgagact tgaagcccag caacctactt 600
ttgaatgcaa attgtgacct caaaatttgt gattttggac ttgctcgtac cacctcagaa 660
accgatttta tggctgagtt tgttgtcaca agatggtata gggcaccgga acttctgttg 720
aattcctctg aatatactgc agcaattgat gtgtggtctg tgggctgtat ttttatggaa 780
ctcatggatc gtaaaccttt gtttcctgga agagatcatg tccatcaatt acgtctacta 840
atggagctca tcggaacgcc aaatgaggct gatctggatt ttgtaaatga aaatgcaaga 900
agatacattc gccaacttcc tagacatgca aggcagtcct ttcctgaaaa atttccacat 960
gttcatcctt tagcaattga tctggttgaa aagatgctga catttgatcc tagacagaga 1020
ataacagttg aaggtgccct tgcacatcct tacctggcat cactgcatga cataagtgat 1080
gagccagtct gctcatcacc cttcagcttt gacttcgagc agcatgcatt gtccgaggaa 1140
caaatgaagg atctaatcta ccaagaaggc cttgcgttca accctgatta ccagtag 1197
<210> 3
<211> 398
<212> PRT
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<400> 3
Met Asp Ala Gly Ala Gln Pro Pro Asp Thr Glu Met Ala Glu Ala Gly
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Gly Gly Gln Gln Pro Pro Ala Ala Ala Ala Ala Ala Gly Ala Gly Ala
20 25 30
Gly Ala Gly Met Met Glu Asn Ile Gln Ala Thr Leu Ser His Gly Gly
35 40 45
Arg Phe Ile Gln Tyr Asn Ile Phe Gly Asn Val Phe Glu Val Thr Ala
50 55 60
Lys Tyr Lys Pro Pro Ile Leu Pro Ile Gly Lys Gly Ala Tyr Gly Ile
65 70 75 80
Val Cys Ser Ala Leu Asn Ser Glu Thr Gly Glu Gln Val Ala Ile Lys
85 90 95
Lys Ile Ala Asn Ala Phe Asp Asn Lys Ile Asp Ala Lys Arg Thr Leu
100 105 110
Arg Glu Ile Lys Leu Leu Arg His Met Asp His Glu Asn Ile Val Ala
115 120 125
Ile Arg Asp Ile Ile Pro Pro Pro Gln Arg Asn Ser Phe Asn Asp Val
130 135 140
Tyr Ile Ala Cys Glu Leu Met Asp Thr Asp Leu His Gln Ile Ile Arg
145 150 155 160
Ser Asn Gln Ala Leu Ser Glu Glu His Cys Gln Tyr Phe Leu Tyr Gln
165 170 175
Ile Leu Arg Gly Leu Lys Tyr Ile His Ser Ala Asn Val Leu His Arg
180 185 190
Asp Leu Lys Pro Ser Asn Leu Leu Leu Asn Ala Asn Cys Asp Leu Lys
195 200 205
Ile Cys Asp Phe Gly Leu Ala Arg Thr Thr Ser Glu Thr Asp Phe Met
210 215 220
Thr Glu Tyr Val Val Thr Arg Trp Tyr Arg Ala Pro Glu Leu Leu Leu
225 230 235 240
Asn Ser Ser Glu Tyr Thr Ala Ala Ile Asp Val Trp Ser Val Gly Cys
245 250 255
Ile Phe Met Glu Leu Met Asp Arg Lys Pro Leu Phe Pro Gly Arg Asp
260 265 270
His Val His Gln Leu Arg Leu Leu Met Glu Leu Ile Gly Thr Pro Asn
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Glu Ala Asp Leu Asp Phe Val Asn Glu Asn Ala Arg Arg Tyr Ile Arg
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Gln Leu Pro Arg His Ala Arg Gln Ser Phe Pro Glu Lys Phe Pro His
305 310 315 320
Val His Pro Leu Ala Ile Asp Leu Val Glu Lys Met Leu Thr Phe Asp
325 330 335
Pro Arg Gln Arg Ile Thr Val Glu Gly Ala Leu Ala His Pro Tyr Leu
340 345 350
Ala Ser Leu His Asp Ile Ser Asp Glu Pro Val Cys Ser Ser Pro Phe
355 360 365
Ser Phe Asp Phe Glu Gln His Ala Leu Ser Glu Glu Gln Met Lys Asp
370 375 380
Leu Ile Tyr Gln Glu Gly Leu Ala Phe Asn Pro Asp Tyr Gln
385 390 395
<210> 4
<211> 1197
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggacgccg gggcgcagcc gccggacacg gagatggcgg aggccggcgg cgggcagcag 60
ccgcctgctg cggctgcggc ggcgggggcg ggggcagggg cggggatgat ggagaacatc 120
caggcgacgc tgagccatgg cgggaggttc atccagtaca acatcttcgg gaacgtgttc 180
gaggtcaccg ccaagtacaa gccccccatc ctccccatcg gcaagggcgc ctacggcatc 240
gtctgctcgg cgctcaactc ggagacgggg gagcaggtgg cgatcaagaa gatcgccaac 300
gcgttcgaca acaagatcga cgccaagcgc acgctcaggg agatcaagct gctccgccac 360
atggaccacg agaatattgt tgccataagg gatatcatac ctcctccaca aaggaattca 420
ttcaatgacg tttatattgc atgtgaattg atggatactg atctgcatca aattattcgc 480
tcaaatcaag cattgtcaga ggagcactgc cagtatttcc tttatcagat tctccgtggc 540
ttgaagtata tacattcagc aaatgtcctt caccgagact tgaagcccag caacctactt 600
ttgaatgcaa attgtgacct caaaatttgt gattttggac ttgctcgtac cacctcagaa 660
accgatttta tgactgagta tgttgtcaca agatggtata gggcaccgga acttctgttg 720
aattcctctg aatatactgc agcaattgat gtgtggtctg tgggctgtat ttttatggaa 780
ctcatggatc gtaaaccttt gtttcctgga agagatcatg tccatcaatt acgtctacta 840
atggagctca tcggaacgcc aaatgaggct gatctggatt ttgtaaatga aaatgcaaga 900
agatacattc gccaacttcc tagacatgca aggcagtcct ttcctgaaaa atttccacat 960
gttcatcctt tagcaattga tctggttgaa aagatgctga catttgatcc tagacagaga 1020
ataacagttg aaggtgccct tgcacatcct tacctggcat cactgcatga cataagtgat 1080
gagccagtct gctcatcacc cttcagcttt gacttcgagc agcatgcatt gtccgaggaa 1140
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<210> 5
<211> 1197
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggacgccg gggcgcagcc gccggacacg gagatggcgg aggccggcgg cgggcagcag 60
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caggcgacgc tgagccatgg cgggaggttc atccagtaca acatcttcgg gaacgtgttc 180
gaggtcaccg ccaagtacaa gccccccatc ctccccatcg gcaagggcgc ctacggcatc 240
gtctgctcgg cgctcaactc ggagacgggg gagcaggtgg cgatcaagaa gatcgccaac 300
gcgttcgaca acaagatcga cgccaagcgc acgctcaggg agatcaagct gctccgccac 360
atggaccacg agaatattgt tgccataagg gatatcatac ctcctccaca aaggaattca 420
ttcaatgacg tttatattgc atatgaattg atggatactg atctgcatca aattattcgc 480
tcaaatcaag cattgtcaga ggagcactgc cagtatttcc tttatcagat tctccgtggc 540
ttgaagtata tacattcagc aaatgtcctt caccgagact tgaagcccag caacctactt 600
ttgaatgcaa attgtgacct caaaatttgt gattttggac ttgctcgtac cacctcagaa 660
accgatttta tgactgagta tgttgtcaca agatggtata gggcaccgga acttctgttg 720
aattcctctg aatatactgc agcaattgat gtgtggtctg tgggctgtat ttttatggaa 780
ctcatggatc gtaaaccttt gtttcctgga agagatcatg tccatcaatt acgtctacta 840
atggagctca tcggaacgcc aaatgaggct gatctggatt ttgtaaatga aaatgcaaga 900
agatacattc gccaacttcc tagacatgca aggcagtcct ttcctgaaaa atttccacat 960
gttcatcctt tagcaattga tctggttgaa aagatgctga catttgatcc tagacagaga 1020
ataacagttg aaggtgccct tgcacatcct tacctggcat cactgcatga cataagtgat 1080
gagccagtct gctcatcacc cttcagcttt gacttcgagc agcatgcatt gtccgaggaa 1140
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<210> 6
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
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ggctgcagga attcaagctt atggacgccg gggcgcagc 39
<210> 7
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
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cagatcagta tccatcaatt cacatgcaat ataaacgtca ttga 44
<210> 8
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gctgagtttg ttgtcacaag atggtat 27
<210> 9
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ccatcttgtg acaacaaact cagccataaa atcggtttct gaggt 45
<210> 10
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ggctgcagga attcaagctt atggacgccg gggcgcagc 39
<210> 11
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cagatcagta tccatcaatt cacatgcaat ataaacgtca ttga 44
<210> 12
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atccagtcac tatggtcgac ctactggtaa tcagggttga a 41
<210> 13
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gaattgatgg atactgatct g 21
<210> 14
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gtccatcaca gtttgccagt 20
<210> 15
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
agatcgttat gtttatcggc act 23
<210> 16
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
agacttgaag cccagcaacc t 21
<210> 17
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
agttccggtg ccctatacca t 21
<210> 18
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
tgctatgtac gtcgccatcc ag 22
<210> 19
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
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aatgagtaac cacgctccgt ca 22
Claims (10)
1.水稻MAPK6基因突变体,其为DN-OsMAPK6,其氨基酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的水稻MAPK6基因突变体,其特征在于,其核苷酸序列如SEQ IDNO.2所示。
3.水稻MAPK6基因突变体,其为CA-OsMAPK6,其氨基酸序列如SEQ ID NO.3所示。
4.如权利要求3所述的水稻MAPK6基因突变体,其特征在于,其核苷酸序列如SEQ IDNO.4所示。
5.含有权利要求2或4所述水稻MAPK6基因突变体的表达载体。
6.含有权利要求5所述表达载体的宿主细胞。
7.权利要求1-2任一所述的水稻MAPK6基因突变体在使水稻籽粒长度、宽度、厚度或/和重量减小中的应用。
8.权利要求3-4任一所述的水稻MAPK6基因突变体在使水稻籽粒长度、宽度、 厚度或/和重量增加中的应用。
9.权利要求1-4任一所述的水稻MAPK6基因突变体在制备转基因植物中的应用。
10.权利要求1-4任一所述的水稻MAPK6基因突变体在农作物改良育种、制种中的应用。
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