阅读说明：本技术 一种龙眼单果重性状调控基因DlCNR8及其蛋白与应用 (Longan single fruit weight character regulatory gene DlCNR8, protein and application thereof ) 是由 决登伟 桑雪莲 石胜友 唐建民 于 2021-01-05 设计创作，主要内容包括：本发明提供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。本发明对DlCNR8基因进行克隆,分析该基因的序列结构、进化关系、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,并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,并转化到Mico Tom番茄中进行功能分析。结果表明DlCNR8基因如番茄FW2.2一样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。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。(The invention provides a longan single fruit weight character regulatory gene DlCNR8, the nucleotide sequence of which is shown in SEQ ID No. 1. The invention is right DlCNR8 The gene is cloned, the sequence structure, the evolutionary relationship and the tissue expression of the gene are analyzed, and a fusion protein expression vector (35S: DlCNR8-GFP) which is transferred into arabidopsis thaliana mesophyll protoplast cells by a PEG mediated method and observed by a laser confocal microscopeSubcellular localization, etc. Meanwhile, an overexpression vector is constructed and transformed into Mico Tom tomato for functional analysis. The results show that DlCNR8 The gene regulates the cell division rate to carry out negative regulation on the fruit weight by acting on downstream protein like tomato FW 2.2. The result not only lays an important foundation for the development of theoretical research on the weight/size of fruit trees such as longan and the like, but also provides important gene resources and molecular markers for the subsequent development of new variety breeding of large-fruit type longan by utilizing molecular-assisted breeding.)

1. A longan single fruit weight character regulatory gene DlCNR8 is characterized in that: the nucleotide sequence is shown in SEQ ID No. 1.

2. The longan single fruit weight trait regulatory gene DlCNR8 expressed expression trait regulatory protein as claimed in claim 1, wherein the amino acid sequence is shown as SEQ ID No. 2.

3. A vector comprising the longan single fruit weight trait-controlling gene according to claim 1.

4. An engineered bacterium comprising the vector of claim 3.

5. The use of the longan single fruit weight trait regulating gene as defined in claim 1 for regulating longan single fruit weight trait.

6. The use of claim 5, wherein: infecting the engineering bacteria of claim 4 on plants to obtain transgenic plants with single fruit weight character regulation.

Technical Field

The invention relates to the technical field of molecular biology, in particular to a longan single fruit weight character regulation main effect QTL and application of a candidate gene DlCNR8 gene thereof in controlling fruit development.

Background

Longan (Dimocarpus longana Lour.) (2n ═ 2x ═ 30) belongs to Sapindaceae (Sapindaceae) longan genus (Dimocarpus) plants, and is an important economic forest in southern areas of China. Widely planted in places such as Guangdong, Fujian, Guangxi, Hainan, Yunnan, Sichuan and Chongqing in China. The cultivation area and the fruit yield of Chinese longan are the first place in the world all the year round. Although China has more than 2000 years of cultivation history, and the longan breeding workers in China carry out longan breeding work from the end of the 80 th 20 th century, the competitiveness of longan in domestic and foreign markets in China is still weak, and the main reason is the shortage of large-fruit high-quality longan varieties. The fundamental reason is the lack of understanding of the genetic mechanism of the single fruit weight character of the longan. Therefore, the genetic mechanism of the single fruit weight of the longan is deeply analyzed, the key genes for regulating and controlling the single fruit weight character of the longan are excavated, and the method has important significance for accelerating the cultivation of new varieties of large-fruit high-quality longan and improving the quality and the efficiency of longan industry in China.

As a complex quantitative trait, single fruit weight is susceptible to genetic background and environment, and there is no clear correspondence between phenotype and genotype (Lubobin. longan microsatellite marker development, breeding application and excellent hybrid line breeding [ D ]. south China agricultural university, 2014.). Therefore, the method is an effective way for analyzing the single fruit weight character during the QTL analysis based on the genetic map. In recent years, with the progress of genetic means, molecular biology technology, bioinformatics platform and the like, some QTL or genes for controlling the weight traits of plant grains or fruits have been found on model plants such as rice, tomato and the like, such as rice An-1, An-2, GN4-1, GW2, qSW5, GS2, GS5, GW8, GS3 and GL7/GW7, and tomato 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, GoZ, Yang Z, Huang G.Characterisation of a noveloritting science, Zhang J, Zhang R, Yi C, Gong Z, Yang Z, Huang G.26, Zhang J, acoustic Q1, Zhang J, S12, Zhang J, 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.Recirculation of the free metabolometer in the tomato weaving [ J ] Cell,2018,172(1): 249-261.). These genes increase or decrease cell number, primarily by regulating the frequency of cell division or cell cycle duration, ultimately affecting yield. Here, Fw2.2 is the first QTL associated with fruit weight cloned from a plant, which is located at the end of chromosome 2 in tomato. The Fw2.2 large fruit allele causes an increase in fruit weight by increasing the Number of cells, resulting in an increase in the fruit placenta and the columella region, which contributes 30% to fruit weight gain (Li Z, He C. physiology floridana Cell Number Regulator1 codes a Cell membrane-attached modulator of Cell and novel control front size [ J ]. Journal of experimental bulk, 2014,66(1):257- > 270.). FW2.2-like (FWL) genes are widely present in animals and plants, and the amino acid sequence similarity of the regulatory factors is generally low, but the regulatory factors contain a PLAC8 structural domain. The FWL gene is involved in various biological processes such as Plant growth and development and Environmental response, and has been studied to confirm that the FWL gene is involved in the uptake and transport of heavy metal ions in plants (Qiao K, Tian Y, Hu Z, Chai T. where Cell Number Regulator CNR10 enzymes, transformation, and accumulation of heavy metals in plants [ J ]. Environmental science & technology,2018,53(2):860-867.), the formation of nodules and the nitrogen fixation process (Qiao Z, Brechnemacher L, Smith B, Strout GW, human W, Taylor C, Russell SD, Stachy G, Libault M.W.GmL 1 (2-2-like) nucleotide C, transformation and Cell Number Regulator J.7. molecular modification J. (Cell Number Regulator J.7) and Cell Number Regulator J.35. Cell No. 7. Cell J.3. 3. Cell No. 7. Cell No. 2. molecular modification J. (Cell No. 2. 3. molecular modification J.: Cell No. 7. 3. Cell No. 7. 3. molecular modification of Plant Cell No. 7. Cell No. 7, 2014,66(1):257 and 270), and the like. Among FWLs that regulate the growth of Plant organs by involving Cell division and changing the Number of cells, the FWL is also called Cell Number Regulator (CNR) (Guo M, Rupe MA, Dieter JA, zuo J, Spielbauer D, Duncan KE, Howard RJ, Hou Z, Simmons cr.cell Number Regulator1 affects Plant and organ size in main: indications for crop yield enhancement and heterosis [ J ] Plant Cell,2010,22(4): 1057.). At present, the research of the CNR participating in the mechanism of regulating the weight of the fruit is only limited in model plants of tomato and berry, and the regulation pathway is not clear. There are few studies on the Fw2.2/CNR gene of woody trees, and only few studies have been reported in pear and avocado (Dahan Y, Rosenfeld R, Zadiranov V, Irishimovitch V.A disposed connected roll for an avacado f 2.2-like gene as a negative regulator of free cell division [ J ]. plant, 2010,232(3):663 ], Jia T, Bin Z, Luo S, Li X, Wu B, Li J.cloning, localization and expression of two of w2.2-like genes in small and large-like spectra [ J ]. internal of agricultural, 2016. Functions of No. 15. Functions are unknown. However, the traditional mapping aspects, such as AFLP and the like, are adopted in the researches, and mapping populations are all less than 100, so that the map precision is not high, and the method cannot be used for accurately and efficiently identifying candidate genes of QTL (quantitative trait locus) segments.

Disclosure of Invention

The invention aims to provide a longan single fruit weight character regulation gene.

The invention also aims to provide the protein for regulating gene expression of the longan single fruit weight character.

The invention also aims to provide application of the longan single fruit weight character regulatory gene.

The purpose of the invention is realized according to the following technical scheme:

a longan single fruit weight character regulatory gene DlCNR8 has a nucleotide sequence shown in SEQ ID No. 1.

A longan single fruit weight character regulation protein has an amino acid sequence shown as SEQ ID No. 2.

The invention also provides a vector containing the coding gene.

The invention also provides an engineering bacterium containing the carrier.

The invention further provides application of the gene in the aspect of regulation and control of the single fruit weight character of longan.

Further, the engineering bacteria are infected into plants to obtain transgenic plants with single fruit weight character regulation.

The invention has the following beneficial effects:

in the early stage, 200 parts of 'pineapple flower' (female parent) × 'big black circle' (male parent) hybrid F1 generation and parent plants are taken as materials, and the materials are sequenced by using an RAD-seq technology and SNP markers are developed to construct a longan high-density genetic map. And (3) performing linkage positioning analysis by combining the single fruit weight data for 2 years continuously, and screening 12 stable QTL sites associated with the single fruit weight. Wherein one gene located in the QTL region of lg10 linkage group encodes a cell number regulator gene: DlCNR8 gene. Fruits in different development stages of large fruit strain FD105 and small fruit strain in F1 generations are selected as materials, Dlo _011045.1(DlCNR8) genes of main effect QTL (qSFW-10-3) are determined to be candidate genes for controlling single fruit weight characters through qRT-PCR analysis, then the full length of ORF of the genes is cloned, and the sequence structure, the evolutionary relationship, the tissue expression condition and the like of the genes are analyzed. qRT-PCR analysis shows that the gene shows differential expression in fruits of different development stages of the large fruit strain FD105 and the small fruit strain FD21 in the F1 generation.

The invention clones the gene DlCNR8, analyzes the sequence structure, evolutionary relationship and tissue expression of the gene, and constructs a fusion protein expression vector (35S: DlCNR8-GFP) is transferred into arabidopsis mesophyll protoplast cells by a PEG mediated method, and the subcellular localization condition of the gene is observed by a laser confocal microscope. Meanwhile, an overexpression vector is constructed and transformed into Mico Tom tomato for functional analysis. The result shows that the gene DlCNR8 contains a conserved domain PLAC8 of a cell number factor regulator (cell number regulator), has closer relation with the CNR8 subfamily member of fruit trees such as citrus and the like, has tissue expression specificity and has the highest expression quantity in young fruits; the subcellular localization result shows that the genes are respectively dotted on the plasma membrane; the expression was significantly up-regulated in the critical fruit development stage of the F1 generation mini-fruit line FD21, 60-80DAP, and the fruits of the over-expressed transgenic line were also significantly smaller than those of wild-type Mico Tom tomato. The above results indicate that the gene DlCNR8 can regulate cell division rate to carry out negative regulation on fruit weight by acting downstream protein like tomato FW 2.2. The result not only lays an important foundation for the development of theoretical research on the weight/size of fruit trees such as longan and the like, but also provides important gene resources and molecular markers for the subsequent development of new variety breeding of large-fruit type longan by utilizing molecular-assisted breeding.

Drawings

FIG. 1: mapping of DlCNR8 in linkage maps.

FIG. 2: longan DlCNR8 gene PCR amplification graph.

FIG. 3: alignment of CNR protein sequences between different species. The box section indicates the amino acid sequence of the PLAC8 domain.

FIG. 4: evolutionary tree analysis chart of similar sequences in longan DlCNR and GenBank.

FIG. 5: relative expression of DlCNR8 in different tissues of longan. Different letter targets indicate that the difference reaches a significant level.

FIG. 6: relative expression profiles of DlCNR8 in fruit development of different F1 progeny.

FIG. 7: pulp weight change plots of FD21 and FD105 at 5 time periods.

FIG. 8: subcellular localization of DlCNR8 protein in arabidopsis mesophyll protoplasts; GFP: green fluorescent protein; chloroplast: chloroplast autofluorescence; bright: bright field; merged: fusing 2 kinds of fluorescence and bright field; the scale bar is 10 μm.

FIG. 9: DlCNR8 transgenic tomato fruit development phenotype map.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

EXAMPLE 1 cloning of the Gene of interest

Materials and methods

1.1 plant Material

Selecting 3 groups of 'season honey' longan with consistent growth vigor and age (9 years) as sampling trees, and taking organs of 'season honey' longan such as flowers, flower buds, leaves, fruit skins, pulps, roots, seeds, stems, young fruits (whole fruits 60 days after flowers) and the like as materials to perform tissue expression analysis. Selecting 3 groups of F1 generation big fruit type plant line FD105 and small fruit type plant line FD21 longan with consistent growth vigor and age (10 years) as sampling trees, and taking longan pulp of 60, 70, 80, 90 and 100 days after flowering as materials for fruit development analysis. All the test devices are repeated for 3 times, and after sampling, the samples are immediately put into liquid nitrogen for quick freezing and are transferred into a refrigerator with the temperature of minus 80 ℃ for storage for later use.

1.2 cloning and bioinformatic analysis of DlCNR8 Gene sequences

The base Sequence and amino acid Sequence information of the DlCNR8 gene (Dlo-011045.1) were obtained from the longan genome database (NCBI Sequence Read Archive, SRA 315202). Primers CNR8-S and CNR8-A (Table 1) were designed from the ORF sequence of the DlCNR8 gene using Primer Premier 5.0, and were synthesized by Shimayihui Biotech Co., Ltd., Guangzhou. The RNA of 'season honey' longan leaves is extracted by using a plant RNA extraction kit of Beijing Huayue biology company, a PrimeScript RT-PCR kit of Takara company is adopted, the specific operation steps refer to an instruction book, and cDNA is reversely transcribed as a template to perform PCR amplification cloning of a DlCNR8 gene. The amplification conditions were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 40s for 35 cycles (denaturation-extension); extending for 10min at 72 ℃, and storing at 4 ℃. And (3) carrying out gel cutting recovery and purification on the amplification product, connecting the amplification product to a pMD18-T vector, transforming DH5 alpha competent cells, carrying out PCR (polymerase chain reaction) screening on positive clones, and selecting positive monoclonals to send to Yihui-Chiyuan biological technology limited company (Guangzhou) for sequencing.

The protein domains were predicted using the online software SMART program (http:// SMART. embedded elberg. de /), and the isoelectric points and molecular weights of the proteins were analyzed using ExPASY (http:// expay. org/tools /). According to the cDNA sequence obtained by cloning, the amino acid sequence is subjected to homology comparison by using BLASTp, and meanwhile, the MEGA 5 software is used for amino acid sequence homology analysis and phylogenetic analysis to construct a Neighbor-Joining evolutionary tree, wherein the number of times of repetition is 1000, and the others are all default settings.

1.3 expression analysis

qRT-PCR primers qCNR8-S and qCNR8-A (Table 1) were designed from the cloned DlCNR8 gene sequence and tested using BLASTn at NCBI to ensure primer specificity. The specific primer sequences are shown in Table 1 by taking an Actin gene (Dlo-028674) of longan as an internal reference gene.

Primer information used in Table 1

Tab.1 Information of primers used

The apparatus used for the qRT-PCR reaction was LightCycler 480 from Roche and the PCR reaction enzyme was SYBR Green Master Mix from Takara. The reaction system was 20mL, in which 40ng of template cDNA, 250nM of each of the upstream and downstream primers, 10. mu.L of SYBR Green Master Mix, and ddH for the remainder₂And (4) supplementing and finishing. Reaction procedure: pre-denaturation at 94 ℃ for 5 min; melting curves were made after 40 cycles at 94 ℃ for 10s, 59 ℃ for 20s, 72 ℃ for 30s (95 → 65 ℃, 0.1 ℃/s). By use of 2^-ΔΔCtThe relative expression level of the DlCNR8 gene was calculated. All samples were run in 3 replicates and negative controls were set. Mean statistics were performed using Excel software, one-way anova with SPSS software was performed to analyze the significance of differences in changes of the gene of interest in different tissues and materials (P < 0.05), and plotted using SigmaPlot 12.5 software.

Example 2 subcellular localization analysis

Primers (terminator removal) were designed based on the cloned DlCNR8 gene sequence (Table 1), and the full ORF length of DlCNR8 was amplified and the PCR reaction procedure was as described above. The PCR product is detected by 1% agarose gel electrophoresis, purified and then connected to a pMD18-T vector to transform DH5 alpha. Single colonies were picked and subjected to PCR detection and sequencing of the upgraded particles. The plasmids pBWA (V), HS-osgfp and DlCNR8 were digested with EcoR I, recovered and enzymatically ligated. Transferring the plasmid after enzyme connection into escherichia coli DH5 alpha, selecting correct strains for sequencing after positive detection, and then extracting to obtain pBWA (V) HS-DlCNR8-osgfp plasmid. Subsequently transferred into Arabidopsis protoplasts by the PEG-mediated method (Yoos D, Cho Y H, Screen J. Arabidopsis lysophyl proto-plates: a versatile cell system for transformed gene expression analysis [ J ] Nature Protocols, 2007, 2 (7): 1565.). Dark culture is carried out at 28 ℃ for 24-48 h, and the observation is carried out by a laser confocal microscope. pBWA (V) HS-osgfp empty was used as a control.

Example 3 construction of overexpression vector and functional verification of transgenic tomato

Using specific PCR primers OECNR8-S/OECNR8-A (Table 1), PCR amplification was performed using longan cDNA as a template. The 5 'end of the primer is respectively added with a BamH I restriction enzyme site, and the 5' end of the reverse primer is respectively added with a Sac I restriction enzyme site. The obtained PCR product was ligated with pMD19-T vector and sequenced. And finally, extracting a plasmid with correct sequencing, performing double digestion on the pBI121 and the plasmid with correct sequencing by using BamH I and Sac I respectively, and constructing a plant expression vector containing a DlCNR8 target gene by using T4 DNA ligase, wherein the plant expression vector is named as pBI121-DlCNR 8. The constructed overexpression vector pBI121-DlCNR8 was transformed into Agrobacterium strain GV3101 by freeze-thawing method with liquid nitrogen (see Arread W, Waheed M T, Mysore K S, et al, Agrobacterium-mediated transformation of tomato with rolB genes in enhancement of fruit quality and yield resistance against tomato growth [ J ] PLoS One, 2014, 9 (5): e96979.) and DlCNR8 gene was transformed into tomato (Micro-Tom) by flower-blossom infection method to obtain seeds of T0 generation. Positive tomatoes are screened on MS solid culture medium containing 30ug/ml, and positive transgenic tomato seedlings are detected by pBI121 plasmid specific primers. Transgenic plants of the T3 generation were grown separately in the same environment as the wild type and compared for their fruit development phenotype.

Example 4 results and analysis

1. Location information of DlCNR8 Gene

In the earlier stage, 200 parts of 'pineapple flower' (female parent) × 'big black circle' (male parent) hybrid F1 generation and parent plants are taken as materials, and the materials are sequenced by using RAD-seq technology and SNP markers are developed to construct a longan high-density genetic map. And (3) performing linkage positioning analysis by combining the single fruit weight data for 2 years continuously, and screening 12 stable QTL sites associated with the single fruit weight. Fruits of large fruit strain FD105 and small fruit strain in F1 generations at different development stages are selected as materials, and Dlo _011045.1(DlCNR8) genes of a main effect QTL (qSFW-10-3) are determined as candidate genes for controlling single fruit weight traits through qRT-PCR analysis. The gene is positioned in the 10 th linkage group of a longan genome, and the specific position information is as follows: scaffold 209: 27358 FIG. 1. with 29541.

2. Cloning and bioinformatics analysis of DlCNR8 Gene

Using longan pulp cDNA as template, a fragment of about 700bp was amplified using CNR8-S/CNR8-A (Table 1) (FIG. 2). And (5) displaying a sequencing result. The size of the gene (Dlo-011045.1) is 732bp, 243 amino acids are coded, the molecular weight is 26.34kDa, and the theoretical isoelectric point is 5.35. It is named as DlCNR8 according to the genetic relationship with other crop CNR family members. Amino acid sequence analysis showed [ sweet orange CsCNR8(Citrus sinensis, XP-006478313.1); crimen pomelo CcCNR8(Citrus clementina, XP — 006441807.2) ], DlCNR8 contains 1 PLAC8 domain and is a member of the CNR family (fig. 3).

The amino acid sequence of DlCNR8 was subjected to homology search using BLASTp, and then a phylogenetic tree was constructed using MEGA 6.0 software (fig. 4). The results show that DlCNR8 is evolutionarily closer to CNR8 members of citrus and grape, classified in the CNR8 subfamily.

3. Tissue expression characteristic analysis of DlCNR8 gene

The qRT-PCR result shows that the DlCNR8 gene is expressed in 9 longan tissues tested, but the expression is tissue-specific, wherein the expression is the highest in young fruits and is about 4 times of that of seeds. Second expression in leaves and pulp (fig. 5).

4. Expression pattern of DlCNR8 gene in flower and fruit development process

Using qRT-PCR technology, we analyzed the expression pattern of DlCNR8 during fruit development of the F1 generation large fruit type strain FD105 and the small fruit type strain FD 21. The results showed that the gene DlCNR8 showed a significant increase in FD21 (FIG. 6) with fruit development at 60-80 days after anthesis. Similar to the change in pulp weight (fig. 7), the expression level of DlCNR8 was up-regulated by 4.1-fold and 10.8-fold at postanthesis 70 and 80d, respectively. While no significant changes occurred during the FD105 fruit development stage. This result suggests that DlCNR8 may be involved in the development of the pulp organs at an early stage.

5. Subcellular localization analysis of DlCNR8 Gene

In order to detect the location of the DlCNR8 protein in cells, a fusion protein expression vector (35S: DlCNR8-GFP) containing enhanced Green Fluorescent Protein (GFP) was constructed in the study, transferred into Arabidopsis mesophyll protoplast cells by a PEG-mediated method, and observed by a confocal laser microscope. As shown in fig. 8, under excitation at 480nm wavelength, 35S: DlCNR8-GFP punctate fluorescence signal only upstream of cytoplasmic and cellular membranes, whereas 35S: the GFP control group showed no clear localization, and GFP signals were observed throughout the cells. This result suggests that the DlCNR8 protein may be localized on the plasma membrane of cells.

6. Phenotypic analysis of tomato transformed with DlCNR8 Gene

Transgenic results showed that tomato plants over-expressing the DlCNR8 gene had smaller fruits and also significantly decreased yields relative to wild type (fig. 9), which indicates that overexpression of the DlCNR8 gene significantly decreased fruit weight, size and yield, and speculated that the longan DlCNR8 gene may negatively regulate plant single fruit weight traits by regulating expression of downstream related genes.

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