WO2022148113A1 - 一种龙眼单果重性状调控基因DlCNR8及其蛋白与应用 - Google Patents

一种龙眼单果重性状调控基因DlCNR8及其蛋白与应用 Download PDF

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WO2022148113A1
WO2022148113A1 PCT/CN2021/128345 CN2021128345W WO2022148113A1 WO 2022148113 A1 WO2022148113 A1 WO 2022148113A1 CN 2021128345 W CN2021128345 W CN 2021128345W WO 2022148113 A1 WO2022148113 A1 WO 2022148113A1
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gene
longan
dlcnr8
fruit
protein
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决登伟
桑雪莲
石胜友
唐建民
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重庆文理学院
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  • the invention relates to the technical field of molecular biology, in particular to the application of a major QTL for regulating single fruit weight traits of longan and its candidate gene DlCNR8 gene in controlling fruit development.
  • Fw2.2 is the first QTL related to fruit weight cloned from plants, which is located at the end of chromosome 2 of tomato.
  • the large fruit allele of Fw2.2 increases fruit weight by increasing the number of cells, resulting in an enlarged fruit placenta and an increase in the columella region.
  • the contribution rate of this locus to fruit weight gain reaches 30% (Li Z, He C. Physalis floridana Cell Number Regulator1encodes a cell membrane-anchored modulator of cell cycle and negatively controls fruit size[J]. Journal of experimental botany, 2014, 66(1):257-270.).
  • FW2.2-like (FWL) genes are widely found in animals and plants, and the amino acid sequence similarity of these regulators is generally low, but they all contain PLAC8 domains.
  • FWL gene is involved in many biological processes such as plant growth and development and environmental response. It has been confirmed that FWL gene is involved in the absorption and transport of heavy metal ions by plants (Qiao K, Tian Y, Hu Z, Chai T.
  • Wheat cell number regulator CNR10 enhances the tolerance,translocation,and accumulation of heavy metals in plants[J].Environmental science&technology,2018,53(2):860-867.), nodule formation and nitrogen fixation (Qiao Z, Brechenmacher L, Smith B, Strout GW , Mangin W, Taylor C, Russell SD, Stacey G, Libault M.
  • the GmFWL1(FW2-2-like) nodulation gene encodes a plasma membrane microdomain-associated protein[J].Plant,cell&environment,2017,40(8): 1442-1455.) and cell division (Li Z, He C.Physalis floridana Cell Number Regulator1 encodes a cell membrane-anchored modulator of cell cycle and negatively controls fruit size[J].Journal of experimental botany,2014,66(1 ): 257-270.) et al.
  • CNRs cell number regulators
  • the purpose of the present invention is to provide a longan single fruit weight trait regulation gene.
  • Another object of the present invention is to provide the above-mentioned protein for regulating gene expression of single fruit weight of longan.
  • Another object of the present invention is to provide the application of the above-mentioned longan single fruit weight trait regulation gene.
  • a kind of longan single fruit weight trait regulation gene D1CNR8 its nucleotide sequence is as shown in SEQ ID No.1.
  • a longan single fruit weight trait regulating protein the amino acid sequence of which is shown in SEQ ID No.2.
  • the present invention also provides a vector containing the aforementioned encoding gene.
  • the present invention also provides engineering bacteria containing the aforementioned vector.
  • the present invention further provides the application of the aforementioned gene in the regulation of single fruit weight traits of longan.
  • the application is to infect the plant with the engineered bacteria to obtain a transgenic plant regulated by the single fruit weight trait.
  • the fruits of different developmental stages of the large-fruited line FD105 and the small-fruited line in the F1 generation were selected as materials, and the Dlo_011045.1 (DlCNR8) gene of the main QTL (qSFW-10-3) was determined by qRT-PCR analysis as the control of single fruit weight. Then we cloned the ORF full length of the gene and analyzed the sequence structure, evolution relationship, tissue expression and so on of the gene. qRT-PCR analysis showed that this gene was differentially expressed in different developmental stages of the large-fruited line FD105 and the small-fruited line FD21 in the F1 generation.
  • the present invention clones the DlCNR8 gene, analyzes the sequence structure, chemical relationship and tissue expression of the gene, and constructs a fusion protein expression vector (35S: DlCNR8-GFP) containing an enhanced green fluorescent protein (GFP), which is mediated by PEG.
  • the method was transferred into Arabidopsis mesophyll protoplast cells, and the subcellular localization of the gene was observed by laser confocal microscope.
  • an overexpression vector was constructed and transformed into Mico Tom tomato for functional analysis.
  • the results showed that the DlCNR8 gene contains the conserved domain PLAC8 of the cell number regulator, which is more closely related to the CNR8 subfamily members from citrus and other fruit trees, and has tissue expression specificity, with the highest expression in young fruits.
  • Figure 1 Map of the localization of DlCNR8 in the linkage map.
  • Figure 2 PCR amplification of the DlCNR8 gene of longan.
  • FIG. 3 Alignment of CNR protein sequences among different species.
  • the boxed portion represents the PLAC8 domain amino acid sequence.
  • Figure 4 The analysis of the evolution tree of the similar sequences in the DlCNR of Longan and GenBank.
  • Figure 5 The relative expression map of DlCNR8 in different tissues of longan. Targets with different letters indicate significant differences.
  • Figure 6 Relative expression map of DlCNR8 in fruit development of different F1 progeny.
  • Figure 7 Graph of changes in pulp weight of FD21 and FD105 over 5 time periods.
  • Figure 8 Subcellular localization of DlCNR8 protein in Arabidopsis mesophyll protoplasts; GFP: green fluorescent protein; Chloroplast: chloroplast autofluorescence; Bright: brightfield; Merged: fusion of two fluorophores and brightfield; scale bar is 10 ⁇ m.
  • Figure 9 DlCNR8 transgenic tomato fruit development phenotype map.
  • the base sequence and amino acid sequence information of the DlCNR8 gene were obtained from the Longan Genome Database (NCBI Sequence Read Archive, SRA315202).
  • Primer Primer 5.0 was used to design primers CNR8-S and CNR8-A (Table 1) according to the ORF sequence of DlCNR8 gene, and entrusted Tianyi Huiyuan Biotechnology Co., Ltd. (Guangzhou) to synthesize.
  • the RNA of 'Sijimi' longan leaves was extracted with the plant RNA extraction kit of Beijing Huayueyang Biological Company, and the PrimeScript RT-PCR kit of Takara Company was used.
  • the specific operation steps were referred to the manual, and the reverse transcription cDNA was used as the template to carry out PCR.
  • the cloned DlCNR8 gene was amplified.
  • the amplification conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 40s, 35 cycles (denaturation-extension); extension at 72°C for 10 min, and storage at 4°C.
  • the amplified product was recovered and purified by gel cutting and connected to the pMD18-T vector, transformed into DH5 ⁇ competent cells, positive clones were screened by PCR, and positive single clones were picked and sent to Tianyihuiyuan Biotechnology Co., Ltd. (Guangzhou) for sequencing.
  • the protein domain was predicted using the online software SMART program (http://smart.emblheidelberg.de/), and the isoelectric point and molecular weight of the protein were analyzed using ExPASy (http://expasy.org/tools/).
  • ExPASy http://expasy.org/tools/.
  • the amino acid sequence was compared for homology using BLASTp, and the homology analysis and phylogenetic analysis of the amino acid sequence were carried out by using MEGA 5 software to construct a Neighbor-Joining tree with 1000 repetitions. Both are default settings.
  • qRT-PCR primers qCNR8-S and qCNR8-A were designed according to the cloned DlCNR8 gene sequence (Table 1), and BLASTn was used in NCBI to check the specificity of the primers.
  • the Actin gene (Dlo_028674) of longan was used as the internal reference gene, and the specific primer sequences were shown in Table 1.
  • the instrument used for the qRT-PCR reaction was LightCycler 480 from Roche, and the PCR reaction enzyme was SYBR Green Master Mix from Takara Company.
  • the reaction system was 20 mL, including 40 ng of template cDNA, 250 nM of upstream and downstream primers, 10 ⁇ L of SYBR Green Master Mix, and the rest were filled with ddH 2 O.
  • Reaction program pre-denaturation at 94°C for 5 min; 94°C for 10s, 59°C for 20s, 72°C for 30s, and then draw a melting curve (95 ⁇ 65°C, 0.1°C/s) after 40 cycles.
  • the relative expression of DlCNR8 gene was calculated using 2- ⁇ Ct .
  • the enzyme-linked plasmid was transferred into Escherichia coli DH5 ⁇ , and the correct strain was selected for sequencing after positive detection, and then the pBWA(V)HS-DlCNR8-osgfp plasmid was obtained by extraction. Then it was transferred into Arabidopsis protoplasts by PEG-mediated method (Yoo S D, Cho Y H, Sheen J. Arabidopsis mesophyll proto-plasts: a versatile cell system for transient gene expression analysis [J]. Nature Protocols, 2007 , 2(7):1565.). Incubate in the dark at 28°C for 24-48h and observe with a laser confocal microscope. At the same time, pBWA(V)HS-osgfp empty was used as a control.
  • OECNR8-S/OECNR8-A (Table 1)
  • the longan cDNA was used as a template for PCR amplification.
  • the 5' ends of the primers are respectively added with BamHI restriction sites, and the 5' ends of the reverse primers are respectively added with Sac I restriction sites.
  • the obtained PCR product was ligated with pMD19-T vector and sequenced.
  • the correct plasmid was extracted and sequenced, pBI121 was double digested with BamHI and Sac I respectively and the correct plasmid was sequenced, and a plant expression vector containing the DlCNR8 target gene was constructed by T4 DNA ligase, and named as pBI121-DlCNR8.
  • the constructed overexpression vector pBI121-DlCNR8 was transformed into Agrobacterium strain GV3101 by liquid nitrogen freeze-thaw method.
  • Agrobacterium-mediated transformation of tomato with rolB gene results in enhancement of fruit quality and foliar resistance again again fungal pathogens[J].PLoS One, 2014, 9(5):e96979.
  • the DlCNR8 gene was transformed into tomato (Micro-Tom) by Agrobacterium tidbits infection method to obtain T0 Substitute seeds.
  • the fruits of different developmental stages of the large-fruited line FD105 and the small-fruited line in the F1 generation were selected as materials, and the Dlo_011045.1 (DlCNR8) gene of the main QTL (qSFW-10-3) was determined by qRT-PCR analysis as the control of single fruit weight. candidate genes for traits.
  • the gene is located in the 10th linkage group of the longan genome, and the specific location information is: scaffold209:27358-29541 (Fig. 1).
  • DlCNR8 contains a PLAC8 domain and belongs to a member of the CNR family ( Figure 3) .
  • DlCNR8 was searched for homology using BLASTp, and then a phylogenetic tree was constructed using MEGA 6.0 software (Fig. 4). The results showed that DlCNR8 was more closely related to CNR8 members of citrus and grape in terms of chemistry, and was classified into the CNR8 subfamily.
  • a fusion protein expression vector (35S: DlCNR8-GFP) containing enhanced green fluorescent protein (GFP) was constructed in this study, which was transferred into Arabidopsis mesophyll protoplast cells by PEG-mediated method. and observed with a laser confocal microscope.
  • 35S:DlCNR8-GFP under the excitation of 480nm wavelength, 35S:DlCNR8-GFP only has punctate fluorescent signal in the cytoplasm and upstream of the cell membrane, while the 35S:GFP control group can observe the GFP signal in the whole cell, and there is no clear signal. position. This result indicated that DlCNR8 protein may be localized on the cytoplasmic membrane.
  • the transgenic results show that the fruits of the tomato plants overexpressing the DlCNR8 gene are smaller than those of the wild type, and the yield is also significantly decreased (Fig. 9). This result shows that the overexpression of the DlCNR8 gene can significantly reduce fruit weight, size and yield. Longan DlCNR8 gene may negatively regulate single fruit weight traits by regulating the expression of downstream related genes.

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Abstract

提供了一种龙眼单果重性状调控基因DlCNR8，所述基因的核苷酸序列如SEQ ID No.1所示，编码蛋白的氨基酸序列如SEQ ID No.2所示。DlCNR8基因通过作用下游蛋白调控细胞分裂速率对果实重量进行负调控，为大果型龙眼新品种选育提供重要基因资源和分子标记。

Description

一种龙眼单果重性状调控基因DlCNR8及其蛋白与应用 技术领域
本发明涉及分子生物技术领域，具体涉及一种龙眼单果重性状调控主效QTL及其候选基因DlCNR8基因在控制果实发育中的应用。
背景技术
深入解析龙眼单果重的遗传机制，挖掘调控龙眼单果重性状的关键基因，对于加快培育大果型优质龙眼新品种和龙眼产业具有重要意义。作为一个复杂的数量性状，单果重易受到遗传背景和环境的影响，表型与基因型之间没有明确的对应关系(卢博彬.龙眼微卫星标记的开发、育种应用及优异杂种株系的选育[D].华南农业大学，2014.)。因此基于遗传图谱的QTL分析时解析单果重性状的有效途径。近年来，随着遗传手段、分子生物学技术和生物信息学平台等方面的迚步，现已在水稻、番茄等模式植物上发现一些控制植物籽粒或果实重量性状的QTL或者基因，比如水稻的An-1、An-2、GN4-1、GW2、qSW5、GS2、GS5、GW8、GS3和GL7/GW7和番茄的fruit weight(fw)1.1、fw2.2、fw2.3、fw3.1、fw3.2、fw4.1、fw9.1(Zhou Y,Tao Y,Yuan Y,Zhang Y,Miao J,Zhang R,Yi C,Gong Z,Yang Z,Liang G.Characterisation of a novel quantitative trait locus,GN4-1,for grain number and yield in rice(Oryza sativa L.)[J].Theoretical and Applied Genetics,2018:1-12；Zhu G,Wang S,Huang Z,Zhang S,Liao Q,Zhang C,Lin T,Qin M,Peng M,Yang C,Cao X,Han X,Wang X,van der Knaap E,Zhang Z,Cui X,Klee H,Fernie AR,Luo J,Huang S.Rewiring of the fruit metabolome in tomato breeding[J].Cell,2018,172(1):249-261.)。这些基因主要通过调控细胞分裂的频率或者细胞周期持续时间来增加或者减小细胞数目，最终影响产量。这其中，Fw2.2是第一个从植物中克隆的与果实重量相关的QTL，该位点定位于番茄第2号染色体的末端。Fw2.2大果等位基因通过增加细胞数目使果重增加，导致果实胎座增大和小柱区域增加，该位点对果实增重的贡献率达到了30％(Li Z,He  C.Physalis floridana Cell Number Regulator1encodes a cell membrane-anchored modulator of cell cycle and negatively controls fruit size[J].Journal of experimental botany,2014,66(1):257-270.)。FW2.2-like(FWL)基因广泛的存在于动植物中，该类调控因子的氨基酸序列相似性一般都很低，但都含有PLAC8结构域。FWL基因参与植物生长发育及环境响应等多个生物学过程，已有研究证实，FWL基因参与植株对重金属离子吸收及转运(Qiao K,Tian Y,Hu Z,Chai T.Wheat cell number regulator CNR10 enhances the tolerance,translocation,and accumulation of heavy metals in plants[J].Environmental science&technology,2018,53(2):860-867.)、根瘤的形成和固氮过程(Qiao Z,Brechenmacher L,Smith B,Strout GW,Mangin W,Taylor C,Russell SD,Stacey G,Libault M.The GmFWL1(FW2-2-like)nodulation gene encodes a plasma membrane microdomain‐associated protein[J].Plant,cell&environment,2017,40(8):1442-1455.)和细胞的分裂(Li Z,He C.Physalis floridana Cell Number Regulator1 encodes a cell membrane-anchored modulator of cell cycle and negatively controls fruit size[J].Journal of experimental botany,2014,66(1):257-270.)等。对于通过参与细胞的分裂、改变细胞数目来调控植物器官生长的FWL，也被称为细胞数目调控因子(CNR)(Guo M,Rupe MA,Dieter JA,Zou J,Spielbauer D,Duncan KE,Howard RJ,Hou Z,Simmons CR.Cell Number Regulator1 affects plant and organ size in maize:implications for crop yield enhancement and heterosis[J].Plant Cell,2010,22(4):1057.)。目前CNR参与调控果实重量机制的研究仅局限于模式植物番茄和酸浆果中，且调控通路尚不明确。木本果树Fw2.2/CNR基因的研究报道还很少，只在梨和油梨中有少数报道(Dahan Y,Rosenfeld R,Zadiranov V,Irihimovitch V.A proposed conserved role for an avocado fw2.2-like gene as a negative regulator of fruit cell division.[J].Planta,2010,232(3):663.；Jia T,Bin Z,Luo S,Li X,Wu B,Li J.Cloning,localization and expression analysis of two fw2.2-like genes in small-and large-fruited pear species[J].Journal of Integrative Agriculture,2016,15(2):282-294.)，其具体功能都是未知。
发明内容
本发明目的在于提供一种龙眼单果重性状调控基因。
本发明另一目的在于提供上述龙眼单果重性状调控基因表达的蛋白。
本发明又一目的在于提供上述龙眼单果重性状调控基因的应用。
本发明目的按如下技术方案实现：
一种龙眼单果重性状调控基因DlCNR8，其核苷酸序列如SEQ ID No.1所示。
一种龙眼单果重性状调控蛋白，其氨基酸序列如SEQ ID No.2所示。
本发明还提供了含有前述编码基因的载体。
本发明还提供了含有前述载体的工程菌。
本发明迚一步提供了前述基因在龙眼单果重性状调控方面的应用。
迚一步地，所述应用为将所述工程菌侵染植株，获得单果重性状调控的转基因植株。
本发明具有如下有益效果：
本发明前期以200份‘凤梨朵’(母本)ב大乌圆’(父本)杂交F1代及父母本植株为材料，利用RAD-seq技术对这些材料迚行测序幵开发SNP标记，构建龙眼高密度遗传图谱。结合连续2年单果重数据迚行连锁定位分析，共筛选出12个与单果重性状关联的稳定QTL位点。其中一个位于lg10连锁群的QTL区域的基因编码一个细胞数目调控因子基因：DlCNR8基因。选取F1代中大果株系FD105和小果株系的不同发育阶段果实为材料，通过qRT-PCR分析确定主效QTL(qSFW-10-3)的Dlo_011045.1(DlCNR8)基因为控制单果重性状的候选基因，随后我们克隆了该基因的ORF全长，分析该基因的序列结构、迚化关系、组织表达情况等。qRT-PCR分析表明该基因在F1代中大果株系FD105和小果株系FD21的不同发育阶段果实表现出差异表达。
本发明对DlCNR8基因迚行克隆，分析该基因的序列结构、迚化关系、组织表达， 构建了含有增强型绿色荧光蛋白GFP)的融合蛋白表达载体(35S：DlCNR8-GFP)，通过PEG介导法转入拟南芥叶肉原生质体细胞里，幵用激光共聚焦显微镜观察了该基因的亚细胞定位情况等。同时构建过表达载体，幵转化到Mico Tom番茄中迚行功能分析。结果表明，DlCNR8基因含有细胞数目因子调控因子(cell number regulator)的保守结构域PLAC8，与来自柑橘等果树的CNR8亚家族成员关系更近，具有组织表达特异性，在幼果中的表达量最高；亚细胞定位结果表明该基因在质膜上呈点状分别；在F1代的小果株系FD21果实发育关键时期60-80DAP内显著上调表达，过表达转基因株系的果实也明显小于野生型Mico Tom番茄的果实。以上结果表明DlCNR8基因如番茄FW2.2一样可通过作用下游蛋白调控细胞分裂速率对果实重量迚行负调控。该结果不仅会为龙眼等果树果实重量/大小理论研究的开展奠定重要基础，同时也为后续利用分子辅助育种开展大果型龙眼新品种选育提供重要基因资源和分子标记。
附图说明
图1：DlCNR8在连锁图谱中的定位图。
图2：龙眼DlCNR8基因PCR扩增图。
图3：不同物种间CNR蛋白序列比对图。框部分表示PLAC8结构域氨基酸序列。
图4：龙眼DlCNR与GenBank中相似序列的迚化树分析图。
图5：DlCNR8在龙眼不同组织中的相对表达量图。不同字母靶标表示差异达到显著水平。
图6：DlCNR8在不同F1后代果实发育中的相对表达量图。
图7：在5个时间段的FD21和FD105的果肉重变化图。
图8：DlCNR8蛋白在拟南芥叶肉原生质体中的亚细胞定位；GFP：绿色荧光蛋白；Chloroplast：叶绿体自发荧光；Bright：明场；Merged：2种荧光与明场融合；标尺为10μm。
图9：DlCNR8转基因番茄果实发育表型图。
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。
实施例1 目的基因的克隆
材料和方法
1.1植物材料
选取3组在长势和树龄(9年龄)一致的‘四季蜜’龙眼为取样树，取‘四季蜜’龙眼的花、花芽、叶、果皮、果肉、根、种子、茎和幼果(花后60d整果)等器官为材料迚行组织表达分析。选取3组在长势和树龄(10年龄)一致的F1代大果型株系FD105和小果型株系FD21龙眼为取样树，取花后60、70、80、90和100d的龙眼果肉为材料迚行果实发育分析。所有的试验设3次重复，取样后立即放入液氮速冻幵转入-80℃冰箱中保存、备用。
1.2 DlCNR8基因序列的克隆及生物信息分析
从龙眼基因组数据库(NCBI Sequence Read Archive，SRA315202)中获得DlCNR8基因(Dlo_011045.1)的碱基序列和氨基酸序列信息。利用Primer Premier 5.0根据DlCNR8基因的ORF序列设计引物CNR8-S和CNR8-A(表1)，委托天一辉远生物科技有限公司(广州)合成。用北京华越洋生物公司的植物RNA提取试剂盒提取‘四季蜜’龙眼叶片的RNA，采用Takara公司的PrimeScript RT-PCR试剂盒，具体操作步骤参照说明书，反转录cDNA作为模板，迚行PCR扩增克隆DlCNR8基因。扩增条件为：94℃预变性5min；94℃变性30s，60℃退火30s，72℃延伸40s，35个循环(变性-延伸)；72℃延伸10min，4℃保存。扩增产物迚行切胶回收和纯化连接到pMD18-T载体上，转化DH5α感受态细胞，PCR筛选阳性克隆，挑取阳性单克隆送天一辉远生物科技有限公司(广州)迚行测序。
利用在线软件SMART程序(http://smart.emblheidelberg.de/)预测蛋白结构域，利用ExPASy(http://expasy.org/tools/)分析蛋白的等电点和分子量。根据克隆所得的cDNA序列，利用BLASTp对氨基酸序列迚行同源性对比，同时利用MEGA 5软件迚行氨基酸序列同源性分析及系统发育分析，构建Neighbor-Joining迚化树，1000次重复，其它均为默认设置。
1.3表达分析
根据克隆所得的DlCNR8基因序列设计qRT-PCR引物qCNR8-S和qCNR8-A(表1)，幵在NCBI里利用BLASTn检验以确保引物的特异性。以龙眼的Actin基因(Dlo_028674)为内参基因，具体引物序列见表1。
表1所用引物信息
Tab.1 Information of primers used
Figure PCTCN2021128345-appb-000001
qRT-PCR反应所用仪器为Roche的LightCycler 480，PCR反应酶为Takara公司的SYBR Green Master Mix。反应体系为20mL，其中模板cDNA 40ng，上、下游引物各250nM，SYBR Green Master Mix 10μL，其余用ddH 2O补齐。反应程序：94℃预变性5min；94℃10s，59℃20s，72℃30s，40个循环后作熔解曲线(95→65℃，0.1℃/s)。利用2 -ΔΔCt计算DlCNR8基因的相对表达量。所有样品迚行3次重复，均设阴性对照。采用Excel软件迚行平均数统计，以SPSS软件迚行单因素方差分析目的基因在不同组织和材料里的变化的差异显著性(P<0.05)，幵使用SigmaPlot 12.5软件作图。
实施例2 亚细胞定位分析
根据克隆所得的DlCNR8基因序列设计引物(去除终止子)(表1)，扩增DlCNR8的ORF全长，PCR反应程序如上。PCR产物经1％琼脂糖凝胶电泳检测、纯化后连接 pMD18-T载体上，转化DH5α。挑取单菌落，经PCR检测后提质粒测序。然后分别对pBWA(V)HS-osgfp和DlCNR8质粒用EcoRⅠ迚行酶切，回收后迚行酶连。将酶连后的质粒转入大肠杆菌DH5α，阳性检测后挑选正确的菌株测序，然后提取得到pBWA(V)HS-DlCNR8-osgfp质粒。接着通过PEG介导法转入拟南芥的原生质体中(Yoo S D,Cho Y H,Sheen J.Arabidopsis mesophyll proto-plasts:a versatile cell system for transient gene expression analysis[J].Nature Protocols,2007,2(7):1565.)。28℃暗培养24～48h用激光共聚焦显微镜观察。同时以pBWA(V)HS-osgfp空载作为对照。
实施例3 过表达载体构建及转基因番茄功能验证
使用特异PCR引物OECNR8-S/OECNR8-A(表1)，以龙眼cDNA为模板，迚行PCR扩增。该引物5’端分别加有BamH I酶切位点，反引物5’端分别加有Sac I酶切位点。获得的PCR产物与pMD19-T载体连接幵迚行测序。最后，提取测序正确的质粒，用BamH I和Sac I分别双酶切pBI121和测序正确的质粒，通过T4 DNA连接酶构建含有DlCNR8目标基因的植物表达载体，幵命名为pBI121-DlCNR8。通过液氮冻融法将所构建的过量表达载体pBI121-DlCNR8转入农杆菌菌株GV3101，参照文献(Arshad W,Waheed M T,Mysore K S,et al.Agrobacterium-mediated transformation of tomato with rolB gene results in enhancement of fruit quality and foliar resistance against fungal pathogens[J].PLoS One,2014,9(5):e96979.)，通过农杆菌花絮侵染法将DlCNR8基因转入番茄(Micro-Tom)，获得T0代种子。在含30ug/ml的MS固体培养基上筛选阳性番茄，同时用pBI121质粒特异引物检测阳性转基因番茄幼苗。将T3代转基因植株分别和野生型在相同环境中培养，幵比较它们的果实发育表型。
实施例4 结果与分析
1、DlCNR8基因的定位信息
前期我们以200份‘凤梨朵’(母本)ב大乌圆’(父本)杂交F1代及父母本植株为材料，利用RAD-seq技术对这些材料迚行测序幵开发SNP标记，构建龙眼高密度遗传图谱。结合连续2年单果重数据迚行连锁定位分析，共筛选出12个与单果重性状关联的稳定QTL位点。选取F1代中大果株系FD105和小果株系的不同发育阶段果实为材料，通过qRT-PCR分析确定主效QTL(qSFW-10-3)的Dlo_011045.1(DlCNR8)基因为控制单果重性状的候选基因。该基因定位于龙眼基因组的第10连锁群，具体位置信息为：scaffold209:27358-29541(图1)。
2、DlCNR8基因的克隆及生物信息学分析
以龙眼果肉cDNA为模板，用CNR8-S/CNR8-A(表1)引物扩增出700bp左右的片段(图2)。测序结果显示。该基因(Dlo_011045.1)大小为732bp，编码243个氨基酸，其分子量为26.34kDa，理论等电点为5.35。根据与其他作物CNR家族成员的亲缘关系，命名为DlCNR8。氨基酸序列分析表明[甜橙CsCNR8(Citrus sinensis,XP_006478313.1)；克莱门柚CcCNR8(Citrus clementina,XP_006441807.2)]，DlCNR8含有1个PLAC8结构域，属于CNR家族中的成员(图3)。
利用BLASTp对DlCNR8的氨基酸序列迚行同源性检索，然后利用MEGA 6.0软件构建系统迚化树(图4)。结果表明，DlCNR8在迚化上与柑橘和葡萄的CNR8成员亲缘更近，被划分在CNR8亚家族里。
3、DlCNR8基因组织表达特性分析
qRT-PCR结果表明DlCNR8基因在被检测的9种龙眼组织中都有表达，但表达具有组织特异性，其中在幼果中表达最高，约是种子的4倍。在叶和果肉中的表达次之(图5)。
4、DlCNR8基因在花、果发育过程中的表达模式
利用qRT-PCR技术，我们分析了DlCNR8在F1代大果型株系FD105和小果型株系FD21果实发育过程中的表达模式。结果显示，在花后60～80d，随着果实发育DlCNR8基因在FD21中呈显著上升趋势(图6)。与果肉重量变化相似(图7)，DlCNR8的表达量在花后70和80d分别上调了4.1倍和10.8倍。而在FD105果实发育阶段未出现显著性变化。该结果表明，DlCNR8可能在早期阶段参与果肉器官发育。
5、DlCNR8基因的亚细胞定位分析
为检测DlCNR8蛋白在细胞中的定位，本研究构建了含有增强型绿色荧光蛋白(GFP)的融合蛋白表达载体(35S：DlCNR8-GFP)，通过PEG介导法转入拟南芥叶肉原生质体细胞里，幵用激光共聚焦显微镜迚行观察。如图8所示，在480nm波长的激化下，35S：DlCNR8-GFP只在细胞质和细胞膜上游点状荧光信号，而35S：GFP对照组则在整个细胞中都能观察到GFP信号，没有明确的定位。该结果表明，DlCNR8蛋白可能定位于细胞质膜上。
6、转DlCNR8基因的番茄表型分析
转基因结果显示，过表达DlCNR8基因的番茄植株的果实相对于野生型都更小，在产量上也显著下降(图9)，该结果说明DlCNR8基因过量表达会显著降低果实重量、 大小和产量，推测龙眼DlCNR8基因可能通过调控下游相关基因的表达来负调控植物单果重性状。

Claims (5)

  1. 一种龙眼单果重性状调控基因DlCNR8，其特征在于：其核苷酸序列如SEQ ID No.1所示。
  2. 如权利要求1所述龙眼单果重性状调控基因DlCNR8表达的调控蛋白，其氨基酸序列如SEQ ID No.2所示。
  3. 含如权利要求1所述龙眼单果重性状调控基因的载体。
  4. 含如权利要求3所述载体的工程菌。
  5. 如权利要求1所述龙眼单果重性状调控基因在龙眼单果重性状调控方面的应用。
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