实施例5,突变体中OsHK1基因的功能互补验证
根据OsRHL1基因的CDS序列信息,设计扩增完整ORF的引物,引物上游序列: AAAGTCGACC CACTAGCCAG ACCATCATCA,下游序列: AAATCTAGAC CACTAGCCAG ACCATCATCA。以秀水63的根部cDNA为模板,用高保真酶PrimerSTAR DNA Polymerase扩增出OsHK1基因的ORF,与 pUCm-T连接,连接产物热激转化大肠杆菌DH5α感受态细胞。测序确定序列正确后的质粒用KpnI、XbaI双酶切后与同样经KpnI、XbaI双酶切的pCAMBIA1300改载体(图4)连接,连接产物热激转化大肠杆菌DH5α感受态细胞。酶切检测正确的质粒通过农杆菌株系EHA105介导的水稻遗传转化体系导入到突变体Oshk1-1中,经过侵染、共培养、筛选具有潮霉素抗性的愈伤、分化、生根、练苗移栽,得到转基因植株。农杆菌(EHA105)介导的水稻遗传转化体系主要应用Hiei等人(1994)报道的方法基础上进行优化。该转基因植株中分离的Oshk1-1突变体对6-BA的响应回复成野生型(图3A),说明Oshk1-1的表型确实是有OsHK1突变引起的。该水稻细胞分裂素介导的侧根发生控制基因OsHK1的核苷酸序列如SEQ ID NO:1所示;该基因编码的蛋白质具有SEQ ID NO:2所示的氨基酸序列。为了检测转基因阳性植株是否超表达了OsHK1基因,采用Trizol法分别提取秀水63和2个转基因回复株系叶片的总RNA(我们构建的回复载体为CaMV 35S组成型表达启动子,如果为转基因阳性植株,那么原本不表达的地上部也会超表达OsHK1基因),逆转录成cDNA。半定量反应时,各样品的cDNA模板量先通过OsActin基因的表达量调成一致(扩增条件:58℃,26个循环),再扩增目的基因。目的基因的上游引物序列为:ACCTATAAGGGCAAGCACAAT;下游引物序列为:ATCCAACGCCTTCCTTTC。目的基因长度为361bp,扩增条件为:55℃,28个循环。半定量RT-PCR结果如图3B,证明了转基因阳性株系中的确超表达了OsHK1基因。pCAMBIA1300改载体为本实验室的改造载体,是以市售的pCAMBIA-1300载体为基本骨架,在多克隆位点插入35S启动子和Nos终止子而得到的增强表达载体。
以上列举的仅是本发明的若干个具体实施例,显然,本发明不限于以上实施例,还可以有许多突变型,即在SEQ ID NO:1中添加、取代、插入或缺失一个或几个核苷酸而生成的突变体、等位基因或衍生物,与SEQ ID NO:1所示的cDNA序列至少有80%同源性的基因序列,用于构建具有水稻侧根发生控制功能的转基因水稻。SEQ ID NO:2所示的蛋白质属于细胞分裂素不敏感功能蛋白,其中进行一个或几个氨基酸替换,插入或缺失所获得的功能类似物。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变型,均应是本发明的保护范围。
<110>宁波大学
<120>水稻侧根发生控制基因OsHK1及编码的蛋白质
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 2994
<212> DNA
<213>水稻侧根发生控制基因OsHK1
<220>
<221> CDS
<400> 1
atggggaagc cggaggcgag gagtgggtgg aggaacgcgg cggcggcggc gtgggtgctg 60
gtggccgtgg cgtgcgcggc gtacatgcac tggcacctgc ggcgggagac catggaccgc 120
gccgaggagc ggctcgtcag catgtgcgag gagcgggcga ggatgctgca ggagcagttc 180
ggcgtcaccg tcaaccacgt ccacgccctc gccatcctca tctccacctt ccatttcgag 240
aagttcccgt ccgccatcga ccaggataca tttgcaaagt acacggcaag gacatcgttt 300
gagcgcccac tgctgaatgg agtggcatat gcgcagcgta tcttccatca cgagagggaa 360
atgtttgaga accagcaggg atggattatg aagacgatga agcgacaggc tgcgcctcca 420
caggatgagt atgccccagt gattttttca caggatacgg tttcctacct tgcacgtatc 480
gacatgatgt ctggggagga ggaccgggaa aacatcttac gtgcaagggc taccggcaaa 540
gctgtgttga caaacccatt caggttgctt gggtcaaacc acttgggggt agttctcact 600
tttgctgtgt atcgccctgg tctagctgct gatgcatcag ttgaggagcg tgtggaagca 660
actgctggtt gtttggttgt tgtggtctca tcaaatcaaa tccctactaa cagatatctt 720
ggtggagctt ttgatgtgga gtcactcgtg gaaaatttgc tgagtaaact tgctggcaat 780
caggatattg tagtgaatgt gtatgatgtc acaaatgctt ctgaacctat ggccatgtat 840
ggtcctcaaa gtccagatgg taaagtgtcc ctcttccatg tgagcacgct tgactttggt 900
gatcctttca gggcgcatga aatgaggtgc aggtataggc aaaagcctcc tctgccatgg 960
tctgctatca ctaatccttt gggaactttt gttatatgga tgcttgtggg gtatataatt 1020
tgtgccgcat ggtctcgcta tgataaagtt tcagaggatt gtagaaagat ggaagagctc 1080
aaaactcaag cggaagctgc tgatgttgct aagtcccagt ttctggcaac tgtatcccat 1140
gagatcagaa cacctatgaa tggcgtcctt gtcgtccagc gatctaaaat tctacctttt 1200
gatgtaggaa tgctcgacat gctcttgggg acagatctga gtatgactca gaaggattat 1260
gctcagacag cccagatgtg tggcagagca ttgataacac tgataaatga tgtacttgat 1320
cgggctaaga ttgaagcagg gaaattggag cttgaggcag tgccatttga cctgcggtct 1380
ctcatggatg atgtgatctc cttattttct tcaaaatcaa gagagaagtg cattgagctt 1440
gcagtgtttg tatgtgatga tgttccaaaa gttgttattg gagatccatg gagatatcga 1500
caaatactga caaatttggt gggaaatgca gtcaaattca cagaacgggg ccatgtattt 1560
gtgcgagtct gcctggctga gaactcaaaa gtggaagcta atcaagtcct taatggaacc 1620
atgaatggga aagacggcaa agttgagact acagctaatg gcgccttcaa tactttgagt 1680
ggttttcaag ccgcagatga gcgcaataac tgggattatt ttaagctgct gctatctgat 1740
aaagagcccc atatggatga gcttgagtgc gacagatctt atcaaaatga ttgtgactgt 1800
gtgaccttga tgataagtat tgaggataca ggtgttggga tcccattaca tgcacaagat 1860
cgtgttttta cgcctttcat gcaggctgat agttcaactt caaggaacta tggtggaact 1920
ggcattggtt taagcatcag caaatgttta gctgaactta tgggcggtca aataagtttc 1980
actagccgtc catttgttgg gagtacattt acattctcag ctgtcctcaa gcgctcatgc 2040
aaagatactt caagtgattc aaagaggagc ttatctgagg cactaccaac tgcctttaag 2100
ggaatgaagg ctattttagt agatgggaga cctgtacgtg gagctgtcac acgatatcac 2160
cttaacaggt tgggaatagt tgttaaagtt gtgaataatt tgagtatggg gcttcagact 2220
ttagctggac aaaatggtgt gaaagagtcc agggagaaac tgtctatgct ttttatagag 2280
agtgacatct ggaggcctga gacagatatc ctattattga atcgtctgca tgagctaaag 2340
aataatggtc aggtgcatga gttgccgaag ctagttcttt tggtaacatc agaagctgac 2400
aaggacagat atggatccgc atttgatatt gtgatgtata aacctataag ggcaagcaca 2460
attgcttctt gccttcaaca gttgctaaaa gtagtgatgc ctgaaaggaa agacaatcaa 2520
aataggccct catttctccg cagcttgctg attgggaaga atatattgat tgtagatgat 2580
aataaagtca atctcagggt tgctgcagct gcgctcaaga aatatggtgc caaagttcat 2640
tgtgttgaaa gcggcaaaga tgcagtctcc ttacttcaac aaccacactg ctttgatgca 2700
tgctttatgg atgttcagat gccagagatg gatgggtttg aggcaactag acaaataagg 2760
caaatggagg taaaagcaaa cgaggaaagg aaggcgttgg atttaatgga gggttcaaca 2820
ttcgtcgagt cccatttgcc tgttctggca atgaccgcgg atgtcattca ggcgacatat 2880
gaagaatgta taaaatcggg aatggatggt tatgtgtcca aacctttcga cgaggagcag 2940
ttataccaag ctgtctccag attagtggtg ggaacgaagg aatcagcggt atga 2994
<210>2
<211>997
<212>PRT
<213>水稻侧根发生控制基因OsHK1编码的蛋白质
<400>2
Met Gly Lys Pro Glu Ala Arg Ser Gly Trp Arg Asn Ala Ala Ala
1 5 10 15
Ala Ala Trp Val Leu Val Ala Val Ala Cys Ala Ala Tyr Met His
20 25 30
Trp His Leu Arg Arg Glu Thr Met Asp Arg Ala Glu Glu Arg Leu
35 40 45
Val Ser Met Cys Glu Glu Arg Ala Arg Met Leu Gln Glu Gln Phe
50 55 60
Gly Val Thr Val Asn His Val His Ala Leu Ala Ile Leu Ile Ser
65 70 75
Thr Phe His Phe Glu Lys Phe Pro Ser Ala Ile Asp Gln Asp Thr
80 85 90
Phe Ala Lys Tyr Thr Ala Arg Thr Ser Phe Glu Arg Pro Leu Leu
95 100 105
Asn Gly Val Ala Tyr Ala Gln Arg Ile Phe His His Glu Arg Glu
110 115 120
Met Phe Glu Asn Gln Gln Gly Trp Ile Met Lys Thr Met Lys Arg
125 130 135
Gln Ala Ala Pro Pro Gln Asp Glu Tyr Ala Pro Val Ile Phe Ser
140 145 150
Gln Asp Thr Val Ser Tyr Leu Ala Arg Ile Asp Met Met Ser Gly
155 160 165
Glu Glu Asp Arg Glu Asn Ile Leu Arg Ala Arg Ala Thr Gly Lys
170 175 180
Ala Val Leu Thr Asn Pro Phe Arg Leu Leu Gly Ser Asn His Leu
185 190 195
Gly Val Val Leu Thr Phe Ala Val Tyr Arg Pro Gly Leu Ala Ala
200 205 210
Asp Ala Ser Val Glu Glu Arg Val Glu Ala Thr Ala Gly Cys Leu
215 220 225
Val Val Val Val Ser Ser Asn Gln Ile Pro Thr Asn Arg Tyr Leu
230 235 240
Gly Gly Ala Phe Asp Val Glu Ser Leu Val Glu Asn Leu Leu Ser
245 250 255
Lys Leu Ala Gly Asn Gln Asp Ile Val Val Asn Val Tyr Asp Val
260 265 270
Thr Asn Ala Ser Glu Pro Met Ala Met Tyr Gly Pro Gln Ser Pro
275 280 285
Asp Gly Lys Val Ser Leu Phe His Val Ser Thr Leu Asp Phe Gly
290 295 300
Asp Pro Phe Arg Ala His Glu Met Arg Cys Arg Tyr Arg Gln Lys
305 310 315
Pro Pro Leu Pro Trp Ser Ala Ile Thr Asn Pro Leu Gly Thr Phe
320 325 330
Val Ile Trp Met Leu Val Gly Tyr Ile Ile Cys Ala Ala Trp Ser
335 340 345
Arg Tyr Asp Lys Val Ser Glu Asp Cys Arg Lys Met Glu Glu Leu
350 355 360
Lys Thr Gln Ala Glu Ala Ala Asp Val Ala Lys Ser Gln Phe Leu
365 370 375
Ala Thr Val Ser His Glu Ile Arg Thr Pro Met Asn Gly Val Leu
380 385 390
Val Val Gln Arg Ser Lys Ile Leu Pro Phe Asp Val Gly Met Leu
395 400 405
Asp Met Leu Leu Gly Thr Asp Leu Ser Met Thr Gln Lys Asp Tyr
410 415 420
Ala Gln Thr Ala Gln Met Cys Gly Arg Ala Leu Ile Thr Leu Ile
425 430 435
Asn Asp Val Leu Asp Arg Ala Lys Ile Glu Ala Gly Lys Leu Glu
440 445 450
Leu Glu Ala Val Pro Phe Asp Leu Arg Ser Leu Met Asp Asp Val
455 460 465
Ile Ser Leu Phe Ser Ser Lys Ser Arg Glu Lys Cys Ile Glu Leu
470 475 480
Ala Val Phe Val Cys Asp Asp Val Pro Lys Val Val Ile Gly Asp
485 490 495
Pro Trp Arg Tyr Arg Gln Ile Leu Thr Asn Leu Val Gly Asn Ala
500 505 510
Val Lys Phe Thr Glu Arg Gly His Val Phe Val Arg Val Cys Leu
515 520 525
Ala Glu Asn Ser Lys Val Glu Ala Asn Gln Val Leu Asn Gly Thr
530 535 540
Met Asn Gly Lys Asp Gly Lys Val Glu Thr Thr Ala Asn Gly Ala
545 550 555
Phe Asn Thr Leu Ser Gly Phe Gln Ala Ala Asp Glu Arg Asn Asn
560 565 570
Trp Asp Tyr Phe Lys Leu Leu Leu Ser Asp Lys Glu Pro His Met
575 580 585
Asp Glu Leu Glu Cys Asp Arg Ser Tyr Gln Asn Asp Cys Asp Cys
590 595 600
Val Thr Leu Met Ile Ser Ile Glu Asp Thr Gly Val Gly Ile Pro
605 610 615
Leu His Ala Gln Asp Arg Val Phe Thr Pro Phe Met Gln Ala Asp
620 625 630
Ser Ser Thr Ser Arg Asn Tyr Gly Gly Thr Gly Ile Gly Leu Ser
635 640 645
Ile Ser Lys Cys Leu Ala Glu Leu Met Gly Gly Gln Ile Ser Phe
650 655 660
Thr Ser Arg Pro Phe Val Gly Ser Thr Phe Thr Phe Ser Ala Val
665 670 675
Leu Lys Arg Ser Cys Lys Asp Thr Ser Ser Asp Ser Lys Arg Ser
680 685 690
Leu Ser Glu Ala Leu Pro Thr Ala Phe Lys Gly Met Lys Ala Ile
695 700 705
Leu Val Asp Gly Arg Pro Val Arg Gly Ala Val Thr Arg Tyr His
710 715 720
Leu Asn Arg Leu Gly Ile Val Val Lys Val Val Asn Asn Leu Ser
725 730 735
Met Gly Leu Gln Thr Leu Ala Gly Gln Asn Gly Val Lys Glu Ser
740 745 750
Arg Glu Lys Leu Ser Met Leu Phe Ile Glu Ser Asp Ile Trp Arg
755 760 765
Pro Glu Thr Asp Ile Leu Leu Leu Asn Arg Leu His Glu Leu Lys
770 775 780
Asn Asn Gly Gln Val His Glu Leu Pro Lys Leu Val Leu Leu Val
785 790 795
Thr Ser Glu Ala Asp Lys Asp Arg Tyr Gly Ser Ala Phe Asp Ile
800 805 810
Val Met Tyr Lys Pro Ile Arg Ala Ser Thr Ile Ala Ser Cys Leu
815 820 825
Gln Gln Leu Leu Lys Val Val Met Pro Glu Arg Lys Asp Asn Gln
830 835 840
Asn Arg Pro Ser Phe Leu Arg Ser Leu Leu Ile Gly Lys Asn Ile
845 850 855
Leu Ile Val Asp Asp Asn Lys Val Asn Leu Arg Val Ala Ala Ala
860 865 870
Ala Leu Lys Lys Tyr Gly Ala Lys Val His Cys Val Glu Ser Gly
875 880 885
Lys Asp Ala Val Ser Leu Leu Gln Gln Pro His Cys Phe Asp Ala
890 895 900
Cys Phe Met Asp Val Gln Met Pro Glu Met Asp Gly Phe Glu Ala
905 910 915
Thr Arg Gln Ile Arg Gln Met Glu Val Lys Ala Asn Glu Glu Arg
920 925 930
Lys Ala Leu Asp Leu Met Glu Gly Ser Thr Phe Val Glu Ser His
935 940 945
Leu Pro Val Leu Ala Met Thr Ala Asp Val Ile Gln Ala Thr Tyr
950 955 960
Glu Glu Cys Ile Lys Ser Gly Met Asp Gly Tyr Val Ser Lys Pro
965 970 975
Phe Asp Glu Glu Gln Leu Tyr Gln Ala Val Ser Arg Leu Val Val
980 985 990
Gly Thr Lys Glu Ser Ala Val
995
<210> 3
<211> 44
<212> DNA
<213>水稻侧根发生控制基因OsHK1的466bp处添加的序列
<220>
<221> CDS
<400>3
ACCTTGCACG TATCGACATG ATGTCTGGGG AGGTGCGATT TCAA 44
<210>4
<211>176
<212>PRT
<213>水稻突变体Oshk1-1编码的蛋白质
<400>4
Met Gly Lys Pro Glu Ala Arg Ser Gly Trp Arg Asn Ala Ala Ala
1 5 10 15
Ala Ala Trp Val Leu Val Ala Val Ala Cys Ala Ala Tyr Met His
20 25 30
Trp His Leu Arg Arg Glu Thr Met Asp Arg Ala Glu Glu Arg Leu
35 40 45
Val Ser Met Cys Glu Glu Arg Ala Arg Met Leu Gln Glu Gln Phe
50 55 60
Gly Val Thr Val Asn His Val His Ala Leu Ala Ile Leu Ile Ser
65 70 75
Thr Phe His Phe Glu Lys Phe Pro Ser Ala Ile Asp Gln Asp Thr
80 85 90
Phe Ala Lys Tyr Thr Ala Arg Thr Ser Phe Glu Arg Pro Leu Leu
95 100 105
Asn Gly Val Ala Tyr Ala Gln Arg Ile Phe His His Glu Arg Glu
110 115 120
Met Phe Glu Asn Gln Gln Gly Trp Ile Met Lys Thr Met Lys Arg
125 130 135
Gln Ala Ala Pro Pro Gln Asp Glu Tyr Ala Pro Val Ile Phe Ser
140 145 150
Gln Asp Thr Val Ser Tyr Leu Ala Arg Ile Asp Met Met Ser Gly
155 160 165
Glu Val Arg Phe Gln Thr Leu His Val Ser Thr
170 175