WO2020221029A1 - 玉米类受体激酶基因ZmRLK7及其应用 - Google Patents

玉米类受体激酶基因ZmRLK7及其应用 Download PDF

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WO2020221029A1
WO2020221029A1 PCT/CN2020/085398 CN2020085398W WO2020221029A1 WO 2020221029 A1 WO2020221029 A1 WO 2020221029A1 CN 2020085398 W CN2020085398 W CN 2020085398W WO 2020221029 A1 WO2020221029 A1 WO 2020221029A1
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zmrlk7
gene
maize
receptor
kinase gene
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French (fr)
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何春梅
汪黎明
王娟
刘铁山
关海英
刘春晓
刘强
董瑞
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山东省农业科学院玉米研究所
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

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  • the invention relates to the corn receptor kinase gene ZmRLK7 and its application, and relates to the field of genetic engineering.
  • Corn is not only an important food crop, but also an important feed and industrial raw material. Cultivating excellent corn varieties and increasing corn yields play a pivotal role in the development of the national economy. The yield of corn depends on the yield per plant and planting density, and the yield per plant can be further divided into kernel number per ear and per kernel weight. Compared with other complex agronomic traits, the heritability of grain traits is high and easy to measure. It is not only a model trait for genetic research, but also an important target trait for genetic improvement. Maize is rich in genetic resources, but the rich natural variation in maize has not been fully explored, and the genetic basis for controlling important agronomic traits has not been clarified in depth. Linkage analysis and genome-wide association analysis are used to discover key genes that affect grain traits. It has important practical and theoretical significance to analyze its function and regulation mechanism, construct relevant regulation network and apply it to breeding research.
  • Overexpression of Mn1 gene increased maize grain yield, while the maize ZmINCW1 gene was transformed into Arabidopsis AtcwINV2 (the ZmINCW1 homologous gene in Arabidopsis) mutants, It can complement its phenotype of reduced thousand-seed weight and increase seed yield.
  • the development of genes related to these yields in maize provides genetic resources for the cultivation of high-yield maize. However, most of the above-mentioned related genes have a good effect on a certain corn trait, and the development of genes that have a good effect on a variety of yield-related traits has high-quality significance for improving breeding efficiency and improving economic benefits.
  • the present invention overcomes the above-mentioned shortcomings of the prior art and provides the corn receptor kinase gene ZmRLK7 and its application.
  • the ZmRLK7 gene is obtained based on the expression quantitative trait locus (eQTL) analysis method, and the expression of the gene Up-regulation of the amount will make the plant short, reducing the biomass and yield; down-regulating the gene expression, the plant will become larger, and the biomass and yield will increase.
  • eQTL quantitative trait locus
  • the nucleotide sequence of the corn receptor kinase gene ZmRLK7 is shown in SEQ1.
  • amino acid sequence encoding the maize receptor kinase gene ZmRLK7 is shown in SEQ2.
  • corn receptor kinase gene ZmRLK7 is located in the regulatory hotspot region of chromosome 7, and is a receptor-like protein kinase gene with rich leucine repeats.
  • the ZmRLK7 gene transgenic Arabidopsis plants are shorter, and the antisense gene Arabidopsis plants are larger.
  • the present invention also provides a recombinant expression vector containing the corn receptor-like kinase gene ZmRLK7.
  • the recombinant expression vector contains a fragment of the maize receptor kinase gene ZmRLK7, and the gene fragment is connected to the vector pCAMBIA3301 through the KpnI restriction site, and the forward and reverse directions are identified, and the maize Ubiquitin promoter drives the forward and reverse directions respectively.
  • the recombinant expression vector is used for overexpression, antisense suppression, RNA interference and CRISPR/Cas9 gene editing of the maize ZmRLK7 gene.
  • the present invention also provides a method for increasing plant height, increasing plant biomass and increasing seed yield, including the following steps:
  • S1 vector construction Amplify the full-length coding region of gene ZmRLK7. After the amplified fragment is sequenced correctly, connect the gene fragment to the vector pCAMBIA3301 through the KpnI restriction site. After identifying the forward and reverse directions, select to obtain the maize Ubiquitin promoter-driven reverse The recombinant plasmid PUbi::Antisense-ZmRLK7 for gene expression;
  • S2 Agrobacterium transformation Transform the recombinant plasmid of reverse gene expression described in step S1 into AGL1 Agrobacterium competent cells;
  • S3 plant transformation transfer the Agrobacterium containing the recombinant plasmid obtained in step S2 to YEP liquid medium to collect the cultured bacteria, and use the flower soaking method to transform the bacteria into Arabidopsis thaliana at the mature stage and harvest the seeds to obtain transgenic T1 Seed generation, or genetic transformation of maize immature embryos mediated by the above-mentioned Agrobacterium to obtain transgenic maize plants.
  • the present invention discloses the sequence of the maize receptor kinase gene ZmRLK7 and its role in regulating plant height, biomass and yield.
  • the gene can be used to regulate the growth and development of Arabidopsis, tobacco, corn and other plants and their comprehensive resistance .
  • the increase in the expression level of this gene makes the plant short and the biomass and yield decrease; the decrease in the expression level of the gene makes the plant larger and the biomass and yield increase.
  • the invention can cultivate a kind of high-yield corn and provide gene resources for further improving the yield of cereal crops.
  • FIG. 1 Gene cloning and vector identification diagram.
  • Figure 2 Agrobacterium transformation identification map.
  • FIG. 3 A process diagram of Agrobacterium-mediated genetic transformation of maize immature embryos.
  • FIG. 4 Identification diagram of transgenic corn.
  • Figure 7 The phenotype statistics of transgenic corn kernels.
  • Figure 8 Phenotype map of transgenic corn plants.
  • Figure 9 Subcellular location map of ZmRLK7 gene.
  • the binary vector for plant transformation used in the experiment is modified pCAMBIA3301, which carries the glufosinate-ammonium resistance gene Bar.
  • pCAMBIA3301 which carries the glufosinate-ammonium resistance gene Bar.
  • RLK7-F 5'- GGTACC TAGTACAAACAATTCATGGGGTCT-3' (SEQ ID No. 3)
  • RLK7-R 5'- GGTACC AAGGCTGCTACAAAAACCGACATT-3' (SEQ ID No. 4) as primers.
  • the full-length coding region of ZmRLK7 was amplified with high-fidelity enzyme, and the underlined part is the restriction enzyme KpnI restriction site.
  • the PCR reaction system is 25 ⁇ l, including the following components: 2 ⁇ High-fidelity Master Mix 12.5 ⁇ l, upstream and downstream primers (concentration 10 ⁇ M) each 1 ⁇ l, cDNA 2 ⁇ l, and sterilized water 8.5 ⁇ l.
  • the PCR program is: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 10 s, annealing at 68°C for 15 s, extension at 72°C for 45 s, and 35 cycles; over extension at 72°C for 5 min.
  • the amplified fragment was sequenced correctly, it was ligated into the vector pCAMBIA3301 through the KpnI restriction site, and the forward and reverse directions were identified.
  • the recombinant plasmids PUbi::Sense-ZmRLK7 and PUbi::Antisense were obtained with maize Ubiquitin promoter driving forward and reverse gene expression. -ZmRLK7.
  • Figure 1 shows the result of digestion of the successfully constructed vector.
  • the Sense-ZmRLK7 is connected when 1.2Kb+ and 3.7Kb+ bands are obtained respectively;
  • the bands of 2.2Kb+1.2Kb+631bp+410bp+and 2.6Kb+2Kb+701bp+520bp+large bands are obtained respectively, it is connected to Antisense-ZmRLK7.
  • the method refers to the competent instructions. Take the Agrobacterium competent cells stored at -70°C and melt them in an ice bath, add 1 ⁇ g of recombinant plasmid to 100 ⁇ l competent cells, mix gently, and after an ice bath for 30 minutes, quickly freeze in liquid nitrogen for 5 min; then place in a 37°C water bath Incubate for 5min, ice bath for 2min; add 800 ⁇ l YEP (10g ⁇ L -1 yeast extract; 10g ⁇ L -1 tryptone; 5g ⁇ L-1 sodium chloride; PH 7.0) liquid medium, and shake on a 28°C shaker Incubate for 2-3h. After collecting the bacteria, spread them on YEP plates containing kanamycin and rifampicin, and invert them in a 28°C incubator for 48-72 hours.
  • the bacteria were resuspended in 200ml 5% sucrose solution (containing 0.02% Silwet L-77) and mixed. Immerse the flower branches in the bacterial solution for 30 seconds and shake them up and down gently.
  • the infected Arabidopsis was cultured in the dark for 24 hours, and then cultured under the light for 3-5 days for the second transformation. The seeds are harvested at the maturity stage to obtain transgenic T1 seeds.
  • Extract DNA from leaf tissues of genetically modified and non-transgenic materials and use the following primers for amplification, Ubi-Forward: 5′-GACTCTAATCATAAAAACCCATCTC-3′ (SEQ ID No. 8) and RLK-Reverse: 5′-AAGCGTGACGCGTCCGA GCTAC-3′ (SEQ ID No. 9) is the primer.
  • the forward primer is a promoter-specific primer
  • the reverse primer is a gene-specific primer.
  • the amplification results are shown in Figure 4.
  • qRT-PCR real-time fluorescent quantitative PCR
  • Bó Lé 7500 Real-Time PCR instrument was used for Real-Time PCR, and Opticon MonitorTM Software was used to analyze the experimental results. The results are shown in Figure 5. The gene is highly expressed in primary roots and in immature embryos 15-25 days after pollination. There is almost no expression in leaves, indicating that this gene plays a key role in grain development.
  • Plant the offspring of transgenic Arabidopsis and maize extract DNA from leaves at the seedling stage for PCR detection, and perform plant and seed/grain phenotype statistics for positive plants.
  • FIG. 7 shows that the grain thickness of transantisense gene maize increased significantly by about 20%, and the 100-kernel weight increased significantly by about 24%.
  • Figure 8 shows that the early growth vigor of the antisense gene maize plant is strong, and the plant is robust. This indicates that the gene expression level is down-regulated, and the size of the corn plant, the thickness of the kernel, and the weight of 100 kernels are increased.
  • the corn seeds are sown in vermiculite, and the buds grow to 1-2cm under the light, and they are transferred to the dark to cultivate for 10-11 days. Cut the middle section of the second leaf into about 1mm filaments, add the prepared enzymolysis solution, and incubate in the dark at room temperature for 4 hours. After the incubation, take it out, filter with a 75 ⁇ m filter, centrifuge at 100xg for 2min, collect the corn protoplasts, wash them twice with buffer solution, incubate on ice for 45min-1h, and check the protoplast status under microscope.

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Abstract

提供了一种玉米类受体激酶基因ZmRLK7及其在调节植株高度、生物量和产量中的应用。将该基因的表达量上调，将使植株矮小，生物量及产量降低；将该基因的表达量下调，将使植株变大，生物量及产量提高。利用该基因可以培育一种高产玉米，为提高禾谷类作物产量提供基因资源。

Description

玉米类受体激酶基因ZmRLK7及其应用 技术领域
本发明涉及玉米类受体激酶基因ZmRLK7及其应用，涉及基因工程领域。
背景技术
玉米既是重要的粮食作物，又是重要的饲料和工业原料，培育优良玉米品种、提高玉米单产在国民经济的发展中占有举足轻重的地位。玉米产量取决于单株产量和种植密度，而单株产量可以进一步剖分为穗粒数和单粒重。相对于其他复杂农艺性状，籽粒性状遗传力高，容易度量，既是遗传研究的模式性状，也是遗传改良的重要目标性状。玉米遗传资源丰富，然而存在于玉米中的丰富自然变异至今并未完全挖掘，控制重要农艺性状的遗传基础也未得到深入的阐明，采用连锁分析与全基因组关联分析方法挖掘影响籽粒性状的关键基因，并解析其功能和调控机制，构建相关的调控网络并应用于育种研究有重要的现实和理论意义。
近年来，通过基因组比较分析或关联分析的方法，水稻中一批参与调控粒型与产量性状的基因陆续被克隆，其分子机制也相继被解析，而玉米中相关基因的研究较少。目前，通过同源克隆的方式，玉米中一些与水稻产量基因同源的基因已经被克隆和报道，并表现出与水稻中基因保守的功能。控制水稻粒型的GS3和GW2基因在玉米中的同源基因已经被克隆，且两个基因与玉米籽粒粒型发育和粒重显著正相关。玉米Mn1和ZmINCW1为水稻灌浆基因OsGIF1的两个同源基因，Mn1基因的过表达增加了玉米籽粒产量，而将玉米ZmINCW1基因转化拟南芥AtcwINV2(拟南芥中ZmINCW1同源基因)突变体，可以互补其千粒重降低的表型，使种子产量增加。对玉米中与这些产量相关的基因的开发为高产玉米的培养提供基因资源。但是上述相关基因多是对某一种玉米性状有较好的影响，而开发对多种产量相关性状均具有良好影响的基因，无论是对提高育种效率，还是对提高经济效益均具有优质意义。
发明内容
本发明克服了上述现有技术的不足，提供玉米类受体激酶基因ZmRLK7及其应用，ZmRLK7基因是基于表达数量性状基因座(expression quantitative trait loci,eQTL)分析方法获得的，将该基因的表达量上调，将使植株矮小，生物量及产量降低；将该基因表达量下调， 植株变大，生物量及产量提高。
玉米类受体激酶基因ZmRLK7，其核苷酸序列如SEQ1所示。
进一步的，编码上述玉米类受体激酶基因ZmRLK7的氨基酸序列如SEQ2所示。
进一步的，上述玉米类受体激酶基因ZmRLK7位于第7染色体的调控热点区域，是一个富亮氨酸重复的类受体蛋白激酶基因。
拟南芥中与野生型相比，转正义ZmRLK7基因拟南芥植株矮小，转反义基因拟南芥植株增大。
本发明还提供含有上述玉米的类受体激酶基因ZmRLK7的重组表达载体。
进一步的，所述重组表达载体包含玉米类受体激酶基因ZmRLK7片段，将所述基因片段通过KpnI酶切位点连接到载体pCAMBIA3301中，鉴定正反向，得到玉米Ubiquitin启动子分别驱动正反向基因表达的重组质粒PUbi::Sense-ZmRLK7和PUbi::Antisense-ZmRLK7。
所述重组表达载体用于玉米ZmRLK7基因的过量表达、反义抑制、RNA干扰和CRISPR/Cas9基因编辑。
上述类受体激酶基因ZmRLK7或上述的重组表达载体在提高植物植株高度、增加植株生物量和提高种子产量中的应用。
上述类受体激酶基因ZmRLK7或上述的重组表达载体在获得高产玉米中的应用。
本发明还提供一种提高植物植株高度、增加植株生物量和提高种子产量的方法，包括如下步骤：
S1载体构建：扩增基因ZmRLK7全长编码区，扩增片段测序正确后，将该基因片段通过KpnI酶切位点连接到载体pCAMBIA3301中，鉴定正反向后选取获得玉米Ubiquitin启动子驱动的反向基因表达的重组质粒PUbi::Antisense-ZmRLK7；
S2农杆菌转化：将步骤S1中所述的反向基因表达的重组质粒转化AGL1农杆菌感受态细胞中；
S3植株转化：将含有步骤S2获得的重组质粒的农杆菌转接到YEP液体培养基中收集培养后的菌体，使用浸花法将菌体转化入拟南芥中成熟期收获种子获得转基因T1代种子，或通 过上述农杆菌介导玉米幼胚遗传转化获得转基因玉米植株。
有益效果：
本发明揭示了玉米类受体激酶基因ZmRLK7的序列及其在调节植株高度、生物量和产量中的作用，利用该基因可以调控拟南芥、烟草、玉米等植物的生长发育及其综合抗性。该基因的表达量上升，使植株矮小，生物量及产量降低；该基因表达量下降，植株变大，生物量及产量提高。利用本发明可以培育一种高产玉米，为进一步为提高禾谷类作物产量提供基因资源。
附图说明
图1基因克隆与载体鉴定图。
图2农杆菌转化鉴定图。
图3农杆菌介导遗传玉米幼胚遗传转化过程图。
图4转基因玉米鉴定图。
图5基因表达模式图。
图6转基因拟南芥表型图。
图7转基因玉米籽粒表型统计图。
图8转基因玉米植株表型图。
图9 ZmRLK7基因亚细胞定位图。
具体实施方式
实施例1 含有类受体激酶基因ZmRLK7的表达载体的构建
1、构建表达载体
实验所用植物转化双元载体为改造的pCAMBIA3301，该质粒携带草铵膦抗性基因Bar。以玉米B73 cDNA为模板，以RLK7-F:5′- GGTACCTAGTACAAACAATTCATGGGGTCT-3′(SEQ ID No.3)和RLK7-R:5′- GGTACCAAGGCTGCTACAAAAACCGACATT-3′(SEQ ID No.4)为引物，利用高保真酶扩增ZmRLK7全长编码区，下划线部分为限制性内切酶KpnI酶切位点。PCR反应体系为25μl，包括如下组分：2×High-fidelity Master Mix 12.5μl，上下 游引物(浓度10μM)各1μl，cDNA 2μl，加灭菌水8.5μl。PCR程序为：98℃预变性1min；98℃变性10s，68℃退火15s，72℃延伸45s，循环35次；72℃过度延伸5min。
扩增片段测序正确后，通过KpnI酶切位点连接到载体pCAMBIA3301中，鉴定正反向，得到玉米Ubiquitin启动子分别驱动正反向基因表达的重组质粒PUbi::Sense-ZmRLK7与PUbi::Antisense-ZmRLK7。
图1为构建成功的载体酶切结果，如图所示，用BamHI、PstI对质粒进行酶切时，分别获得1.2Kb+大带和3.7Kb+大带的条带时即为连入Sense-ZmRLK7；分别获得2.2Kb+1.2Kb+631bp+410bp+大带和2.6Kb+2Kb+701bp+520bp+大带的条带时即为连入Antisense-ZmRLK7。
2、转化农杆菌感受态
将以上重组质粒转化AGL1农杆菌感受态细胞，方法参考感受态说明书。取-70℃保存的农杆菌感受态细胞于冰浴中融化，向100μl感受态细胞中加入1μg重组质粒，轻轻混匀，冰浴30min后，液氮速冻5min；然后置于37℃水浴中孵育5min，冰浴2min；加入800μl YEP(10g·L -1酵母膏；10g·L -1胰蛋白胨；5g·L-1氯化钠；PH 7.0)液体培养基，并于28℃摇床上振荡培养2-3h。收集菌体后，涂布于含有卡那霉素和利福平的YEP平板上，于28℃培养箱中倒置培养48-72h。
培养72h后，挑单克隆摇菌提质粒，分别用引物UBQ1 Forward:CTTTTTGTTCGCTTGGTTGTGATGA(SEQ ID No.5)；Sense-ZmRLK7:TCGCAAGCATTGGGATTGTAGAG(SEQ ID No.6)Antisense-ZmRLK7:AAGCGTGACGCGTCCGAGCTAC(SEQ ID No.7)进行PCR鉴定，鉴定结果如图2所示。阳性质粒(P)与Sense-ZmRLK7(1、2泳道)分别扩增出509bp大小条带；阳性质粒(P)Antisense-ZmRLK7(3、4泳道)分别扩增出777bp大小条带。表明农杆菌转化成功。
实施例3 ZmRLK7基因在拟南芥和玉米中的遗传转化
1、浸花法转化拟南芥
将含有重组质粒的农杆菌转接到200ml YEP液体培养基中，待OD600＝0.8～1.2时，5000 r·min -1，离心10min收集菌体。菌体用200ml 5％蔗糖溶液(含0.02％Silwet L-77)重悬，混匀。将花枝浸入菌液中30s，轻轻上下摇动。将侵染后的拟南芥置于黑暗中培养24h，然后置于光下正常培养3-5d，进行第二次转化。成熟期收获种子，即得到转基因T1代种子。
2、农杆菌介导遗传玉米幼胚遗传转化
将含有重组质粒的农杆菌转接到200ml YEP液体培养基中，待OD600＝0.8～1.2时，5000r·min -1，离心10min收集菌体。用授粉以后1.5-2mm的HiII玉米幼胚为受体，进行遗传转化。转化时间5分钟，然后放在诱导培养基上共培养，7天后放在筛选培养基上直至长出抗性愈伤组织。将抗性愈伤组织转入再生培养基，再生植株进行移栽并自交结实。转化过程如图3所示。
3、转基因材料的分子检测结果
3.1 PCR鉴定
取转基因和非转基因材料的叶片组织提取DNA，用如下引物进行扩增，Ubi-Forward:5′-GACTCTAATCATAAAAACCCATCTC-3′(SEQ ID No.8)和RLK-Reverse:5′-AAGCGTGACGCGTCCGA GCTAC-3′(SEQ ID No.9)为引物。正向引物为启动子特异引物，反向引物为基因特异引物,扩增结果如图4所示，非转基因对照(WT)中扩增不出条带，而转基因(泳道1-19)和阳性质粒(P)中能够扩增出目的带，也有假阳性植株如Sense-ZmRLK7扩增结果泳道17。
3.2基因表达模式分析
取玉米不同发育时期的组织，液氮速冻，用Trizol试剂盒提取RNA，然后使用随机六引物进行反转录，获得单链cDNA。以玉米内源基因Actin为内参基因，进行实时荧光定量PCR(qRT-PCR)。使用伯乐7500 Real-Time PCR仪进行Real-Time PCR，Opticon MonitorTM Software对实验结果进行分析。结果如图5所示，该基因在初生根中和授粉后15-25天的幼胚中高表达，在叶片中几乎没有表达说明该基因籽粒发育过程中起关键作用。
4、转基因材料后代性状鉴定
种植转基因拟南芥和玉米后代，在苗期取叶片提取DNA进行PCR检测，阳性植株进行 植株和种子/籽粒进行表型统计。
拟南芥表型结果如图6所示，结果显示，与野生型相比，转正义ZmRLK7基因拟南芥植株矮小，转反义基因拟南芥植株增大。由于转基因拟南芥中结果证明该基因为负调控基因，因此在玉米中仅转化反义基因结构进行功能验证。
玉米表型结果如图7和图8所示，与非转基因对照相比，图7可见转反义基因玉米的籽粒粒厚显著增加，提高约20％，百粒重显著增加，提高约24％；图8可见转反义基因玉米植株早期生长势较强，植株健壮。由此表明所该基因表达水平下调，增加玉米植株的大小及籽粒厚度、百粒重。
5、玉米原生质体转化及基因的亚细胞定位
将ZmRLK7与GFP基因构建融合表达载体，大提质粒备用。玉米种子播于蛭石中，光下出芽长至1-2cm，转至暗处培养10-11天。取第二片叶中段切成约1mm的细丝，加入配制好的酶解液，于室温暗处孵育4h。孵育结束后取出，用75μm滤网过滤，100ⅹg离心2min，收集玉米原生质体，用缓冲溶液稍洗两遍，冰上孵育45min～1h，期间镜检原生质体状态。重悬原生质体并调节浓度至～10-5个细胞/100μl。每个转化实验，各取20μg质粒，加入100μl原生质体细胞，轻弹混匀，加入PEG溶液充分混匀，于28℃暗处静置15min。离心收集并重悬细胞，转移至细胞培养板，28℃培养箱避光培养12-16h。用荧光显微镜镜检，照相。
ZmRLK7蛋白的亚细胞定位在细胞膜和核膜上如图9所示(浅亮色部分为该基因荧光标记的定位)，说明该基因起作用的位置在膜上，这与该基因的二级结构分析中有一个跨膜结构域和一个保守的丝氨酸/苏氨酸激酶结构域结果一致。

Claims (9)

  1. 玉米类受体激酶基因ZmRLK7，其特征在于，核苷酸序列如SEQ1所示。
  2. 如权利要求1所述的玉米类受体激酶基因ZmRLK7，其特征在于，编码所述玉米类受体激酶基因ZmRLK7的氨基酸序列如SEQ2所示。
  3. 如权利要求1所述的玉米类受体激酶基因ZmRLK7，其特征在于，所述玉米类受体激酶基因ZmRLK7位于第7染色体的调控热点区域，是一个富亮氨酸重复的类受体蛋白激酶基因。
  4. 含有如权利要求1所述的玉米的类受体激酶基因ZmRLK7的重组表达载体。
  5. 如权利要求4所述的重组表达载体，其特征在于，将玉米类受体激酶基因ZmRLK7片段通过KpnI酶切位点连接到载体pCAMBIA3301中，鉴定正反向后，得到玉米Ubiquitin启动子分别驱动正反向基因表达的重组质粒PUbi::Sense-ZmRLK7和PUbi::Antisense-ZmRLK7。
  6. 如权利要求4或5所述的重组表达载体，其特征在于，所述重组表达载体用于玉米ZmRLK7基因的过量表达、反义抑制、RNA干扰和CRISPR/Cas9基因编辑。
  7. 权利要求1-3任一项所述的类受体激酶基因ZmRLK7或权利要求4或5所述的重组表达载体在提高植物植株高度、增加植株生物量和提高种子产量中的应用。
  8. 权利要求1-3任一项所述的类受体激酶基因ZmRLK7或权利要求4或5所述的重组表达载体在获得高产玉米中的应用。
  9. 一种提高植物植株高度、增加植株生物量和提高种子产量的方法，其特征在于，包括如下步骤：
    S1载体构建：扩增如权利要求1所述的基因的全长编码区序列，扩增片段测序正确后，将该基因片段通过KpnI酶切位点连接到载体pCAMBIA3301中，鉴定正反向后选取如权利要求5所述的反向基因表达的重组质粒PUbi::Antisense-ZmRLK7；
    S2农杆菌转化：将步骤S1中所述的反向基因表达的重组质粒转化AGL1农杆菌感受态细胞中；
    S3植株转化：将步骤S2获得的含有重组质粒的农杆菌转接到YEP液体培养基中收集培 养后的菌体，使用浸花法将菌体转化入拟南芥中成熟期收获种子获得转基因T1代种子，或通过上述农杆菌介导玉米幼胚遗传转化获得转基因玉米植株。
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