CN114456242A - Prp蛋白及其编码基因和应用 - Google Patents
Prp蛋白及其编码基因和应用 Download PDFInfo
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- CN114456242A CN114456242A CN202210056264.3A CN202210056264A CN114456242A CN 114456242 A CN114456242 A CN 114456242A CN 202210056264 A CN202210056264 A CN 202210056264A CN 114456242 A CN114456242 A CN 114456242A
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Abstract
本发明公开了一种PRP蛋白及其编码基因和应用,PRP蛋白的氨基酸序列如SEQ ID NO.2所示;编码基因的核苷酸序列如SEQ ID NO.1所示。本发明的PRP蛋白可以提高植物C4光合循环速率、增加光合效率,通过导入所述PRP蛋白的编码基因至受体植物,或通过与含有所述编码基因的同种植物有性杂交,将上述编码基因导入受体植物中,得到光合效率或生物量高于所述受体植物的转基因植物。
Description
技术领域
本发明涉及植物生物技术领域,尤其涉及一种PRP蛋白及其编码基因和应用。
背景技术
随人口数量及建设用地面积的不断增加,进一步提高作物单产成为解决未来粮食危机的唯一途径。而实践证明,选育产量潜力比现有品种更高的作物品种是持续增加粮食单产的基础。光合作用是作物产量形成的基础,近些年来通过改良作物光合特性进而提高叶片光能利用率以达提高作物产量的方法正越来越受到各国研究人员的重视。相较于C3植物,C4植物具有CO2浓缩机制,光补偿点高,CO2补偿点低,光呼吸弱,尤其在高温、干旱等逆境条件下表现出比C3植物更高的光合效率。因此将C4植物光合特性导入小麦、水稻等C3作物,培育高光效、高产新品种,对维护世界粮食安全具有十分重要的理论和实践意义。
在小麦、水稻等C3作物中引入C4光合途径,一直是国内外光合作用相关研究的重要研究目标。而在C4光合途径中,磷酸丙酮酸双激酶(PPDK)催化丙酮酸(Pyr)生成磷酸烯醇式丙酮酸(PEP),是C4光合途径中的关键限速步骤之一。如何对C4光合循环步骤中的关键限速酶进行优化设计与改造,是系统生物学及合成生物学研究的前沿问题,而相关功能基因与调控基因等的发掘与利用是此相关研究的关键。
发明内容
本发明要解决的技术问题是克服现有技术的不足,提供一种提高植物C4光合循环速率、增加光合效率的蛋白及其编码基因,以及该蛋白及其编码基因在培育高光效转基因植物中的应用。
为了实现上述目的,本发明提供了一种PRP蛋白,所述PRP蛋白为以下A1~A2中的一种:
A1:如SEQ ID NO.2所示的氨基酸序列;
A2:SEQ ID NO.2所示氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且可提高植物光合效率的由所述A1衍生的蛋白质。
基于一个总的技术构思,本发明还提供了一种上述PRP蛋白的编码基因,所述编码基因为以下B1~B4中的一种:
B1、如SEQ ID NO.1所示的核苷酸序列;
B2、编码序列表中序列2蛋白质序列的多核苷酸序列;
B3、在高严谨条件下可与SEQ ID NO.1第391-1191位限定的DNA序列杂交的核苷酸序列;
B4、与所述B1、B2或B3限定的DNA序列具有90%以上同源性,且编码相同功能蛋白质的DNA序列。
进一步的,所述高严谨条件可为用6×SSC,0.5%SDS的溶液,在65℃下杂交,然后用2×SSC,0.1%SDS和1×SSC,0.1%SDS各洗膜一次。
基于一个总的技术构思,本发明还提供了一种上述PRP蛋白在提高植物C4光合循环速率和增加光合效率中的应用。
上述的应用,进一步的,所述应用的方法为:通过导入所述PRP蛋白的编码基因至受体植物,或通过与含有所述编码基因的同种植物有性杂交,将上述编码基因导入受体植物中,得到光合效率或生物量高于所述受体植物的转基因植物。
上述的应用,进一步的,所述受体植物为以下C1~C4中的一种:
C1、含有C4光合途径PPDK基因的受体植物;
C2、含有PPDK和PEPC基因的受体植物;
C3、含有磷酸烯醇式丙酮合成酶基因的受体植物;
C4、含有PPDK、PEPC和磷酸烯醇式丙酮合成酶基因的受体植物。
上述的应用,进一步的,所述应用的方法为:
S1、通过PCR方法克隆南荻MlPRP基因获得PCR产物;
S2、将所述PCR产物连接到表达载体上得到重组载体;
S3、将所述重组载体转化至大肠杆菌中,提取阳性克隆中的质粒;
S4、将所述质粒通过农杆菌转化获得水稻转化工程菌;
S5、将所述水稻转化工程菌与受体植物的愈伤组织共培养获得转MlPRP基因水稻。
上述的应用,进一步的,所述应用还包括:
S6、将所述转MlPRP基因水稻与同背景转ZmPEPC+ZmPPDK基因水稻进行去雄杂交获得F1代植株;筛选F1代中ZmPEPC、ZmPPDK、MlPRP三基因都存在的单株收种;继续繁殖,于后代植株中筛选三基因聚合纯合株系。
上述的应用,进一步的,所述S1中所述PCR方法所采用的引物包括MlPRP-F和MlPRP-R。所述的MlPRP-F的核苷酸序列如SEQ ID NO.3所示;所述MlPRP-R的核苷酸序列如SEQ ID NO.4所示。
上述的应用,进一步的,所述S2中使用所述基因构建重组表达载体时,在其转录起始核苷酸前可加上任何一种组成型、增强型、诱导型或组织特异型的启动子,所述启动子可单独使用或与其它的启动子结合使用。
上述的应用,进一步的,所述S2中所述表达载体使用GUS基因、GFP基因、萤光素酶基因、庆大霉素抗性基因、卡那霉素抗性基因、潮霉素抗性基因或除草剂抗性基因标记。
上述的应用,进一步的,所述组成型的启动子为Ubi Promoter或CaMV35SPromoter。
所述诱导性型的启动子为光诱导表达启动子,所述光诱导表达启动子为rbcS或Cab。
所述组织特异型的启动子为绿色组织特异性表达启动子,具体为LP2或Rca。
上述的应用,进一步的,所述潮霉素抗性基因为Hyg基因或HPT基因;
所述萤光素酶基因为GFP或GUS基因;
所述除草剂抗性基因为bar基因;
所述庆大霉素抗性基因为aacC1基因。
现有技术相比,本发明的优点在于:
(1)本发明提供了一种PRP蛋白,可以调控C4光合循环关键酶PPDK的磷酸化水平,提高C4光合途径羧化底物磷酸烯醇式丙酮酸(PEP)的再生速率,并最终提高受体植物的光合效率。为提高C4光合循环效率,培育高光效植物新品种提供了新的技术路线。
(2)本发明提供了一种PRP蛋白在提高植物C4光合循环速率和增加光合效率中的应用,通过导入所述PRP蛋白的编码基因至受体植物,或通过与含有所述编码基因的同种植物有性杂交,将上述编码基因导入受体植物中,从而提高植物的光合效率或生物量。
附图说明
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整的描述。
图1为表达载体pSB130-Ubi::PRP结构图。抗性基因为Hyg基因,能使转基因受体获得潮霉素抗性;CaMV35S Promoter为来源于花椰菜花叶病毒的启动子,具有较强的启动活性;Ubi Promoter为玉米泛素基因启动子,在植物中具有较好的启动活性;PRP为南荻的PPDK活性调节蛋白基因MlPRP的CDS序列。
图2为转MlPRP基因再生植株的PCR检测电泳图。M:Marker;1:质粒对照,2-14:T0代水稻植株。
图3为ZmPEPC+ZmPPDK基因与MlPRP基因聚合水稻PCR检测电泳图。M:Marker;1-24:杂交聚合水稻植株。
图4为转基因水稻PPDK磷酸化水平检测结果。A为PPDK蛋白磷酸化与非磷酸化蛋白Western检测图;B为转基因水稻叶片总蛋白SDS-PAGE电泳检测图;C为磷酸化PPDK相对表达量;D为PPDK蛋白相对表达量。其中KC表示转ZmPEPC+ZmPPDK基因水稻;PKC表示转MlPRP与转ZmPEPC+ZmPPDK基因基因聚合水稻。
图5为转基因阳性植株长势图。KC:转ZmPEPC+ZmPPDK基因水稻;P:转MlPRP基因水稻;PKC:ZmPEPC+ZmPPDK基因与MlPRP基因聚合水稻;WT:野生型水稻,湘恢299。
图6为转基因水稻地上部生物量。A为单株叶片总重量;B为单株叶鞘总重量;C为单株茎杆总重量;D为单株地上部总重量。其中KC表示转ZmPEPC+ZmPPDK基因水稻;P表示转MlPRP基因水稻;PKC表示转MlPRP与转ZmPEPC+ZmPPDK基因基因聚合水稻;WT表示野生型水稻,湘恢299。
具体实施方式
以下结合具体优选的实施例对本发明作进一步描述,但并不因此而限制本发明的保护范围。
实施例
以下实施例中所采用的材料和仪器均为市售。
实施例1
一种提高植物C4光合循环速率、增加光合效率的蛋白,名称为PRP(PPDK/PEPSRegulation Protein,PRP),来源于南荻(Miscanthus lutarioriparia L.Liu exRenvoize&S.L.Chen)。
其编码基因的核苷酸序列如SEQ ID NO.1所示,具体为:
atgattgggtgcgccaagccgctcgcggcgccgctgcagccgccgtcccccgccggtcgccgcctcgccccattgttctgcgcccctgactcctctccagctctcacccgcgccgtcgagagcccaggccagtctcagtctgacgtcgccccgccaccacgcccggatgaggtcgcctcgtccctcgcgcggcgagcgagcccgcagctcagtcggtggtcccgcttgcggacgctgcggtcgaaccgccggcctggactggaccgctcggtgctctcctcggcctcgtcctcggcctcggcgccgccggtgaccaagacgtcgcggcccgaggacgctgcggtggcggtggaggatggcgaggacgacgtcgtgtcgaacgggaagtccatctacatagtgtcggatgggaccgggtggaccgcggagcactcggtgagcgccgcgctcggacagttcgagcactgcttcgtcgaccgcggctgtgctgtcaacacccacctcttctccatgattaacgacatggatcgaattcttgaggtaataaagcaagcagcaaaggaaggggcactggttctatatacccttgctgatccttcaatggccgagtcagctaagaaggcctgcgatttctggggtgttccatccactgatgttcttcggcctactgttgaagccattgcttctcatatgggtgttgctccatctggaattccacgaagctctcctagtcgaccgtgtcagctaacagaggattactttcgacggattgatgctatcgattttaccatcaaacaagatgatggggcacagccagagaacctcaaccgtgcagacattgtacttgccggcgtttcacgtacagggaagacaccattgtcaatatatctagcccaaaagggatacaaggtagcaaatgtcccaattgtgatgggagtgaatcttccaaaatccctttttgagatcagccaagacaagatttttggattgacaataaacccagtggttcttcaagcaattagaaagactagggccaaagctctaggttttggtgatgggcagagcaattatgctgaaatggatcatgtgaggcaggaactactccatgcaaatcaaatttttgctcaaaatgcaatgtggccagtcattgcggtcactggaaaagctatagaggaaacagctgctgtcgttgtgaggatttaccttgacaggaaacagaagtactccatgccacgcatatcaaaacgatactag。
SEQ ID NO.1由1254个核苷酸组成,其开放阅读框架(ORF)为自5'末端第1-1254位核苷酸,编码SEQ ID NO.2所示的蛋白质。
在本发明的一个实施例中,编码PRP蛋白的基因还可以是如下1)至3)中任一所述的DNA分子:
1)编码序列表中SEQ ID NO.2蛋白质序列的多核苷酸序列。
2)在高严谨条件下可与序列表中SEQ ID NO.1的第391-1191位限定的DNA序列杂交的核苷酸序列。上述高严谨条件可为用6×SSC,0.5%SDS的溶液,在65℃下杂交,然后用2×SSC,0.1%SDS和1×SSC,0.1%SDS各洗膜一次。
3)与SEQ ID NO.1或上述1)或2)限定的DNA序列具有90%以上同源性,且编码相同功能蛋白质的DNA序列。
其氨基酸序列由417个氨基酸残基组成,如SEQ ID NO.2所示,具体为:MIGCAKPLAAPLQPPSPAGRRLAPLFCAPDSSPALTRAVESPGQSQSDVAPPPRPDEVASSLARRASPQLSRWSRLRTLRSNRRPGLDRSVLSSASSSASAPPVTKTSRPEDAAVAVEDGEDDVVSNGKSIYIVSDGTGWTAEHSVSAALGQFEHCFVDRGCAVNTHLFSMINDMDRILEVIKQAAKEGALVLYTLADPSMAESAKKACDFWGVPSTDVLRPTVEAIASHMGVAPSGIPRSSPSRPCQLTEDYFRRIDAIDFTIKQDDGAQPENLNRADIVLAGVSRTGKTPLSIYLAQKGYKVANVPIVMGVNLPKSLFEISQDKIFGLTINPVVLQAIRKTRAKALGFGDGQSNYAEMDHVRQELLHANQIFAQNAMWPVIAVTGKAIEETAAVVVRIYLDRKQKYSMPRISKRY。
基于一个总的技术构思,SEQ ID NO.2所示氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且可提高植物光合效率的由1)衍生的蛋白质也可以达到与实施例1的蛋白相同或相似的技术效果。
实施例2
一种PRP蛋白,为了便于PRP蛋白的纯化,在由SEQ ID NO.2所示氨基酸残基序列组成的蛋白质的氨基末端或羧基末端连接上Poly-Arg标签。Poly-Arg标签包括5-6个碱基(通常5个),具体为:RRRRR。
实施例3
一种PRP蛋白,为了便于PRP蛋白的纯化,在由SEQ ID NO.2所示氨基酸残基序列组成的蛋白质的氨基末端或羧基末端连接上Poly-His标签。Poly-His标签包括2-10个氨基酸(通常6个),具体为:HHHHHH。
实施例4
一种PRP蛋白,为了便于PRP蛋白的纯化,在由SEQ ID NO.2所示氨基酸残基序列组成的蛋白质的氨基末端或羧基末端连接上FLAG标签。FLAG标签包括8个氨基酸,具体为:DYKDDDDK。
实施例5
一种PRP蛋白,为了便于PRP蛋白的纯化,在由SEQ ID NO.2所示氨基酸残基序列组成的蛋白质的氨基末端或羧基末端连接上Strep-tagⅡ标签。Strep-tagⅡ标签包括8个氨基酸,具体为:WSHPQFEK。
实施例6
一种PRP蛋白,为了便于PRP蛋白的纯化,在由SEQ ID NO.2所示氨基酸残基序列组成的蛋白质的氨基末端或羧基末端连接上C-myc标签。C-myc标签包括8个氨基酸,具体为:EQKLISEEDL。
上述实施例1至实施例6中的PRP蛋白可人工合成,也可先合成其编码基因,再进行生物表达得到。
实施例7
一种实施例1的PRP蛋白在提高植物C4光合循环速率和增加光合效率中的应用,是将上述实施例1至6中任一项所述蛋白的编码基因导入受体植物,或通过与含有所述编码基因的同种植物有性杂交,将上述编码基因导入受体植物中,得到光合效率或生物量高于所述受体植物的转基因植物。受体植物含有C4光合途径PPDK或PPDK和PEPC基因,和/或磷酸烯醇式丙酮合成酶基因(PEPS)。
具体在本实施例中,应用方法包括以下步骤:
(1)南荻MlPRP基因的克隆:采用本领域常用的TRIzol试剂法提取南荻叶片总RNA。
具体步骤为:
1.1、使用Thermo的RevertAid First Strand cDNA Synthesis Kit(FermentasCode:K1622)试剂盒合成第一链cDNA,逆转录反应步骤参照说明书进行。
1.2、设计引物:
MlPRP-F:5’-atgattgggtgcgccaagcc-3’(SEQ ID NO.3);
MlPRP-R:5’-ctagtatcgttttgatatgcg tggcatgg-3’(SEQ ID NO.4)。
1.3、按照以下组份配制PCR反应体系(50μL)
1.4、按以下反应程序进行PCR扩增
PCR产物经1.0%的琼脂糖凝胶电泳检测和切胶回收纯化后,将回收片段TA克隆至pEASY-Blunt Zero Vector(购自全式金公司),转化大肠杆菌、涂平板后,挑取单菌落测序,序列如序列1所示。
(2)载体构建:
2.1、将Sma I限制性内切酶对pSB130载体进行线性化酶切。
2.2、设计同源重组引物PCR克隆Ubi启动子和MlPRP基因编码序列;通过琼脂糖凝胶电泳酶切及PCR扩增产物并切胶回收纯化。
所采用的引物为:
Ubi-cF:gactctagaggatcccctgcagtgcagcgtgacccggt(SEQ ID NO.5);
Ubi-cR:AGCTTGGGtgcagaagtaacaccaaacaacagggtgagca(SEQ ID NO.6);
PRP-cF:ttctgcaCCCAAGCTTGGGATGATTGGGTGC(SEQ ID NO.7);
PRP-cR:tcgagctcggtacccCTAGTATCGTTTTGATATGCGTGGCATGGAGTACTTCT(SEQ IDNO.8)。
2.3、利用全式金公司pEASY-Uni Seamless Cloning and Assembly Kit对线性化载体及PCR扩增片段进行同源重组得到重组载体。
重组载体具体可为图1所示载体。图1所示的表达载体的载体骨架为pSB130,在载体骨架中通过同源重组方法插入玉米Ubi基因启动子和外源基因,外源基因由玉米Ubi基因启动子启动。
(3)转化:将重组载体转化大肠杆菌DH5α感受态细胞,用PCR鉴定正确的单菌落扩大培养并提取质粒。测序验证正确的pSB130-Ubi::PRP质粒如图1所示。
(4)制备水稻工程菌:取测序验证正确的pSB130-Ubi::PRP质粒转化农杆菌EHA105感受态细胞,将PCR检测正确的阳性单克隆菌落扩大培养,保存菌种并用作水稻转化工程菌。
(5)转MlPRP基因水稻的获得:
利用湘恢299水稻种子成熟胚诱导愈伤组织,挑选发育旺盛的愈伤组织继代培养14天后,与水稻转化工程菌共培养3天;将共培养后的愈伤组织转移到选择培养基上暗培养;待愈伤组织发育出鲜黄色颗粒型的抗性愈伤后,将其转移至选择分化培养基光培养;待抗性愈伤分化出绿苗后,转移到生根培养基中继续光照培养;待抗性苗生根,经炼苗后定植到网室中,常规水肥管理至成熟后收获种子,经PCR鉴定获得转MlPRP基因水稻。
图2为转MlPRP基因再生植株的PCR检测电泳图。M:Marker;1:质粒对照,2-14:T0代水稻植株。
对比例1:
一种转ZmPEPC+ZmPPDK基因水稻,按照实施例7的方法,将ZmPEPC+ZmPPDK基因转入至水稻中,获得转ZmPEPC+ZmPPDK基因水稻。
实施例8:
一种实施例1的PRP蛋白在提高植物C4光合循环速率和增加光合效率中的应用,所述应用方法包括以下步骤:
步骤(1)至(4)与实施例7一致。
步骤(5)为:ZmPEPC+ZmPPDK+MlPRP三基因聚合水稻的获得:
将对比例1中同背景转ZmPEPC+ZmPPDK基因水稻和转MlPRP基因水稻种植于网室中,常规水肥管理;20%至30%的水稻植株抽穗时,选择天气晴好的上午进行去雄杂交;收获杂交种后,继续温室种植F1代植株,提取叶片DNA,通过PCR分别检测F1代ZmPEPC、ZmPPDK、MlPRP基因,图3为ZmPEPC+ZmPPDK与MlPRP基因三聚合水稻PCR检测电泳图。图中M:Marker;1-24:杂交聚合水稻植株。通过PCR检测电泳结果去除假杂种、筛选三基因都存在的单株收种;连续检测F2、F3代单株ZmPEPC、ZmPPDK、MlPRP基因聚合情况,筛选三基因聚合纯合株系;分单株收获纯合株系单株种子,获得三基因聚合水稻纯系。
实验一:PPDK蛋白磷酸化及PPDK酶活性检测
(1)PPDK蛋白磷酸化水平检测
在ZmPPDK蛋白第527位苏氨酸附近选取一段多肽链序列LTERGGMTSH,分别合成以T527磷酸化修饰和无磷酸化修饰的多肽链作为抗原,分别免疫新西兰大白兔,获得phosT527和T527抗体。利用phosT527和T527抗体分别对实施例7的转MlPRP基因水稻、对比例1的转ZmPEPC+ZmPPDK基因水稻以及实施例8的三基因聚合水稻的叶片中ZmPPDK蛋白进行Western Blot检测,用ImageJ软件对检测条带进行灰度分析,计算磷酸化蛋白相对含量。
结果参见图4:图4中A为PPDK蛋白磷酸化与非磷酸化蛋白Western检测图;图4中B为转基因水稻叶片总蛋白SDS-PAGE电泳检测图;图4中C为磷酸化PPDK相对表达量。其中KC表示转ZmPEPC+ZmPPDK基因水稻;PKC表示转MlPRP与转ZmPEPC+ZmPPDK基因基因聚合水稻。从图5的结果发现:相较于转ZmPEPC+ZmPPDK基因水稻,ZmPEPC+ZmPPDK与MlPRP三基因聚合水稻叶片ZmPPDK蛋白磷酸化水平显著降低(图4中A,图4中C),说明南荻来源的MlPRP可以调控ZmPPDK蛋白的去磷酸化。
(2)PPDK酶活性检测
PPDK酶活性采用酶偶联反应法进行测定,按照以下配方配制蛋白提取液:50mM pH7.5Tris-HCl,1mM MgCl2,5mM DTT,0.1%Triton X-100(v/v),10%甘油(v/v),5%不溶性PVP粉末(w/v);NaOH调节pH至7.5。将配制好的蛋白提取液4℃保存备用。
按照以下配方配制预反应液:10mM 6-磷酸葡萄糖(G6P),100mM Hepes,20mMMgCl2,0.4mM NADH,4mM丙酮酸,2.5mM ATP,5mM KH2PO4,10mM NH4Cl,20mM NaHCO3,KOH调节pH至8.0,再加入20mM NaHCO3,3U/mL的MDH酶。
分别取100mg实施例7的转MlPRP基因水稻、对比例1的转ZmPEPC+ZmPPDK基因水稻以及实施例8的三基因聚合水稻的叶片,加入1ml预冷的蛋白提取液,在冰浴中研磨,随后转入2ml离心管,13000rpm,4℃离心10min,吸取上清液置于冰上待测。预热酶标仪半小时以上,设定测定波长为340nm。配制200μL反应体系:100μL预反应液;10μL蛋白提取上清液,90μL ddH2O;混匀反应体系后,立即用酶标仪读取OD340的值,每隔30s读取一次,持续读数10min;以每分钟光密度下降0.01为一个酶活单位(U)计算PPDK酶活性。
图4中D为PPDK蛋白相对表达量。从图中结果发现:相较于转ZmPEPC+ZmPPDK基因水稻,ZmPEPC+ZmPPDK与MlPRP三基因聚合水稻叶片ZmPPDK蛋白酶活性显著提高。说明MlPRP通过调控ZmPPDK蛋白的去磷酸化,可以显著提高PPDK酶活性。
实验二:水稻地上部生物量检测
随机区组种植获得的实施例7的转MlPRP基因水稻、对比例1的转ZmPEPC+ZmPPDK基因水稻、实施例8的ZmPEPC+ZmPPDK与MlPRP三基因聚合水稻以及野生型R299,每个基因型3次生物学重复。水稻成熟后每小区取5株分蘖数与平均分蘖较为一致的植株,整个地上部分取回,将叶片、叶鞘、茎及穗分离,烘干后电子天平分别测量叶、鞘、茎和穗的质量,地上生物量为四者相加。
图5为转基因阳性植株长势图。KC:转ZmPEPC+ZmPPDK基因水稻;P:转MlPRP基因水稻;PKC:ZmPEPC+ZmPPDK基因与MlPRP基因聚合水稻;WT:野生型水稻,湘恢299。从图中可以看出:四种转基因水稻的长势良好。证明将本发明所提供的PRP基因导入野生型水稻湘恢299中,获得能够稳定遗传的转PRP基因水稻。
图6为转基因水稻地上部生物量。A为单株叶片总重量;B为单株叶鞘总重量;C为单株茎杆总重量;D为单株地上部总重量。其中KC表示转ZmPEPC+ZmPPDK基因水稻;P表示转MlPRP基因水稻;PKC表示转MlPRP与转ZmPEPC+ZmPPDK基因基因聚合水稻;WT表示野生型水稻,湘恢299。结果显示:相较于其它三组水稻,ZmPEPC+ZmPPDK与MlPRP三基因聚合水稻生物量显著型增加。可以预见MlPRP基因可能通过改善转ZmPEPC+ZmPPDK基因水稻光合效率,提高其干物质积累,并最终增加地上部总生物量。
综合上述结果可知:将本发明所提供的PRP基因导入野生型水稻湘恢299中,获得能够稳定遗传的转PRP基因水稻;在正常生长状态下,转PRP基因水稻植株光合效率明显高于空载体对照植株。说明本发明对培育高光效水稻材料具有重要的理论及应用价值。
以上所述,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制。虽然本发明已以较佳实施例揭示如上,然而并非用以限定本发明。任何熟悉本领域的技术人员,在不脱离本发明的精神实质和技术方案的情况下,都可利用上述揭示的方法和技术内容对本发明技术方案做出许多可能的变动和修饰,或修改为等同变化的等效实施例。因此,凡是未脱离本发明技术方案的内容,依据本发明的技术实质对以上实施例所做的任何简单修改、等同替换、等效变化及修饰,均仍属于本发明技术方案保护的范围内。
序列表
<110> 湖南杂交水稻研究中心
<120> PRP蛋白及其编码基因和应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1254
<212> DNA
<213> 南荻(Miscanthus lutarioriparius)
<400> 1
atgattgggt gcgccaagcc gctcgcggcg ccgctgcagc cgccgtcccc cgccggtcgc 60
cgcctcgccc cattgttctg cgcccctgac tcctctccag ctctcacccg cgccgtcgag 120
agcccaggcc agtctcagtc tgacgtcgcc ccgccaccac gcccggatga ggtcgcctcg 180
tccctcgcgc ggcgagcgag cccgcagctc agtcggtggt cccgcttgcg gacgctgcgg 240
tcgaaccgcc ggcctggact ggaccgctcg gtgctctcct cggcctcgtc ctcggcctcg 300
gcgccgccgg tgaccaagac gtcgcggccc gaggacgctg cggtggcggt ggaggatggc 360
gaggacgacg tcgtgtcgaa cgggaagtcc atctacatag tgtcggatgg gaccgggtgg 420
accgcggagc actcggtgag cgccgcgctc ggacagttcg agcactgctt cgtcgaccgc 480
ggctgtgctg tcaacaccca cctcttctcc atgattaacg acatggatcg aattcttgag 540
gtaataaagc aagcagcaaa ggaaggggca ctggttctat atacccttgc tgatccttca 600
atggccgagt cagctaagaa ggcctgcgat ttctggggtg ttccatccac tgatgttctt 660
cggcctactg ttgaagccat tgcttctcat atgggtgttg ctccatctgg aattccacga 720
agctctccta gtcgaccgtg tcagctaaca gaggattact ttcgacggat tgatgctatc 780
gattttacca tcaaacaaga tgatggggca cagccagaga acctcaaccg tgcagacatt 840
gtacttgccg gcgtttcacg tacagggaag acaccattgt caatatatct agcccaaaag 900
ggatacaagg tagcaaatgt cccaattgtg atgggagtga atcttccaaa atcccttttt 960
gagatcagcc aagacaagat ttttggattg acaataaacc cagtggttct tcaagcaatt 1020
agaaagacta gggccaaagc tctaggtttt ggtgatgggc agagcaatta tgctgaaatg 1080
gatcatgtga ggcaggaact actccatgca aatcaaattt ttgctcaaaa tgcaatgtgg 1140
ccagtcattg cggtcactgg aaaagctata gaggaaacag ctgctgtcgt tgtgaggatt 1200
taccttgaca ggaaacagaa gtactccatg ccacgcatat caaaacgata ctag 1254
<210> 2
<211> 417
<212> PRT
<213> 南荻(Miscanthus lutarioriparius)
<400> 2
Met Ile Gly Cys Ala Lys Pro Leu Ala Ala Pro Leu Gln Pro Pro Ser
1 5 10 15
Pro Ala Gly Arg Arg Leu Ala Pro Leu Phe Cys Ala Pro Asp Ser Ser
20 25 30
Pro Ala Leu Thr Arg Ala Val Glu Ser Pro Gly Gln Ser Gln Ser Asp
35 40 45
Val Ala Pro Pro Pro Arg Pro Asp Glu Val Ala Ser Ser Leu Ala Arg
50 55 60
Arg Ala Ser Pro Gln Leu Ser Arg Trp Ser Arg Leu Arg Thr Leu Arg
65 70 75 80
Ser Asn Arg Arg Pro Gly Leu Asp Arg Ser Val Leu Ser Ser Ala Ser
85 90 95
Ser Ser Ala Ser Ala Pro Pro Val Thr Lys Thr Ser Arg Pro Glu Asp
100 105 110
Ala Ala Val Ala Val Glu Asp Gly Glu Asp Asp Val Val Ser Asn Gly
115 120 125
Lys Ser Ile Tyr Ile Val Ser Asp Gly Thr Gly Trp Thr Ala Glu His
130 135 140
Ser Val Ser Ala Ala Leu Gly Gln Phe Glu His Cys Phe Val Asp Arg
145 150 155 160
Gly Cys Ala Val Asn Thr His Leu Phe Ser Met Ile Asn Asp Met Asp
165 170 175
Arg Ile Leu Glu Val Ile Lys Gln Ala Ala Lys Glu Gly Ala Leu Val
180 185 190
Leu Tyr Thr Leu Ala Asp Pro Ser Met Ala Glu Ser Ala Lys Lys Ala
195 200 205
Cys Asp Phe Trp Gly Val Pro Ser Thr Asp Val Leu Arg Pro Thr Val
210 215 220
Glu Ala Ile Ala Ser His Met Gly Val Ala Pro Ser Gly Ile Pro Arg
225 230 235 240
Ser Ser Pro Ser Arg Pro Cys Gln Leu Thr Glu Asp Tyr Phe Arg Arg
245 250 255
Ile Asp Ala Ile Asp Phe Thr Ile Lys Gln Asp Asp Gly Ala Gln Pro
260 265 270
Glu Asn Leu Asn Arg Ala Asp Ile Val Leu Ala Gly Val Ser Arg Thr
275 280 285
Gly Lys Thr Pro Leu Ser Ile Tyr Leu Ala Gln Lys Gly Tyr Lys Val
290 295 300
Ala Asn Val Pro Ile Val Met Gly Val Asn Leu Pro Lys Ser Leu Phe
305 310 315 320
Glu Ile Ser Gln Asp Lys Ile Phe Gly Leu Thr Ile Asn Pro Val Val
325 330 335
Leu Gln Ala Ile Arg Lys Thr Arg Ala Lys Ala Leu Gly Phe Gly Asp
340 345 350
Gly Gln Ser Asn Tyr Ala Glu Met Asp His Val Arg Gln Glu Leu Leu
355 360 365
His Ala Asn Gln Ile Phe Ala Gln Asn Ala Met Trp Pro Val Ile Ala
370 375 380
Val Thr Gly Lys Ala Ile Glu Glu Thr Ala Ala Val Val Val Arg Ile
385 390 395 400
Tyr Leu Asp Arg Lys Gln Lys Tyr Ser Met Pro Arg Ile Ser Lys Arg
405 410 415
Tyr
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgattgggt gcgccaagcc 20
<210> 4
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ctagtatcgt tttgatatgc gtggcatgg 29
<210> 5
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gactctagag gatcccctgc agtgcagcgt gacccggt 38
<210> 6
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agcttgggtg cagaagtaac accaaacaac agggtgagca 40
<210> 7
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ttctgcaccc aagcttggga tgattgggtg c 31
<210> 8
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tcgagctcgg tacccctagt atcgttttga tatgcgtggc atggagtact tct 53
Claims (10)
1.一种PRP蛋白,其特征在于,所述PRP蛋白为以下A1~A2中的一种:
A1:如SEQ ID NO.2所示的氨基酸序列;
A2:SEQ ID NO.2所示氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且可提高植物光合效率的由所述A1衍生的蛋白质。
2.根据权利要求1所述的PRP蛋白,其特征在于,在所述PRP蛋白的氨基末端或羧基末端连接Poly-Arg标签、Poly-His标签、FLAG标签、Strep-tagⅡ标签或C-myc标签。
3.一种权利要求1或2所述PRP蛋白的编码基因,其特征在于,所述编码基因为以下B1~B4中的一种:
B1、如SEQ ID NO.1所示的核苷酸序列;
B2、编码序列表中序列2蛋白质序列的多核苷酸序列;
B3、在高严谨条件下可与SEQ ID NO.1第391-1191位限定的DNA序列杂交的核苷酸序列;
B4、与所述B1、B2或B3限定的DNA序列具有90%以上同源性,且编码相同功能蛋白质的DNA序列。
4.一种权利要求1或2所述PRP蛋白在提高植物C4光合循环速率和增加光合效率中的应用,其特征在于,所述应用的方法为:通过导入所述PRP蛋白的编码基因至受体植物,或通过与含有所述编码基因的同种植物有性杂交,将上述编码基因导入受体植物中,得到光合效率或生物量高于所述受体植物的转基因植物。
5.根据权利要求4所述的应用,其特征在于,所述受体植物为以下C1~C4中的一种:
C1、含有C4光合途径PPDK基因的受体植物;
C2、含有PPDK和PEPC基因的受体植物;
C3、含有磷酸烯醇式丙酮合成酶基因的受体植物;
C4、含有PPDK、PEPC和磷酸烯醇式丙酮合成酶基因的受体植物。
6.根据权利要求4所述的应用,其特征在于,所述应用的方法为:
S1、通过PCR方法克隆南荻MlPRP基因获得PCR产物;
S2、将所述PCR产物连接到表达载体上得到重组载体;
S3、将所述重组载体转化至大肠杆菌中,提取阳性克隆中的质粒;
S4、将所述质粒通过农杆菌转化获得水稻转化工程菌;
S5、将所述水稻转化工程菌与受体植物的愈伤组织共培养获得转MlPRP基因水稻。
7.根据权利要求6所述的应用,其特征在于,所述应用还包括:
S6、将所述转MlPRP基因水稻与同背景转ZmPEPC+ZmPPDK基因水稻进行去雄杂交获得F1代植株;筛选F1代中ZmPEPC、ZmPPDK、MlPRP三基因都存在的单株收种;继续繁殖,于后代植株中筛选三基因聚合纯合株系。
8.根据权利要求6所述的应用,其特征在于,所述S1中所述PCR方法所采用的引物包括MlPRP-F和MlPRP-R;所述的MlPRP-F的核苷酸序列如SEQ ID NO.3所示;所述MlPRP-R的核苷酸序列如SEQ ID NO.4所示;
和/或,所述S2中使用所述基因构建重组表达载体时,在其转录起始核苷酸前可加上任何一种组成型、增强型、诱导型或组织特异型的启动子,所述启动子可单独使用或与其它的启动子结合使用;
和/或,所述S2中所述表达载体使用GUS基因、GFP基因、萤光素酶基因、庆大霉素抗性基因、卡那霉素抗性基因或庆大霉素抗性基因、卡那霉素抗性基因、潮霉素抗性基因或除草剂抗性基因标记。
9.根据权利要求8所述的应用,其特征在于,
所述组成型的启动子为Ubi Promoter或CaMV35S Promoter;
所述诱导性型的启动子为rbcS或Cab;
所述组织特异型的启动子LP2或Rca。
10.根据权利要求8所述的应用,其特征在于,所述潮霉素抗性基因为Hyg基因或HPT基因;所述萤光素酶基因为GFP或GUS基因;所述除草剂抗性基因为bar基因;所述庆大霉素抗性基因为aacC1基因。
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