阅读说明：本技术 小麦产量杂种优势相关调控基因TalncRNA1809及其应用 (Wheat yield heterosis related regulatory gene TalncRNA1809 and application thereof ) 是由 高世庆 赵昌平 刘永杰 公杰 刘钰涵 张风廷 唐益苗 陈兆波 于 2020-05-18 设计创作，主要内容包括：本发明属于农业生物领域,具体涉及小麦产量杂种优势相关基因TalncRNA1809和应用。该基因的核苷酸序列如SEQ ID NO.1所示,对改良提高产量、加速高产分子育种进程具有十分重要的理论和实际意义。(The invention belongs to the field of agricultural biology, and particularly relates to a gene TalncRNA1809 related to wheat yield and heterosis and application thereof. The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the gene has very important theoretical and practical significance for improving the yield and accelerating the breeding process of high-yield molecules.)

1. Plant yield heterosis related gene TalncRNA1809 is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. A recombinant expression vector comprising the plant yield heterosis associated gene TalncRNA1809 of claim 1.

3. A recombinant strain comprising the plant yield heterosis associated gene TalncRNA1809 of claim 1.

4. Use of the plant yield heterosis related gene TalncRNA1809 according to claim 1.

5. Use of the plant yield heterosis related gene TalncRNA1809 according to claim 1 for increasing plant yield.

6. A method for increasing plant yield, comprising the step of overexpressing in a plant the plant yield heterosis-related gene TalncRNA1809 according to claim 1.

7. The method of claim 6, wherein said plant comprises wheat, rice, corn, cucumber, tomato, poplar, turf grass or alfalfa.

Technical Field

The invention belongs to the field of agricultural biology, and particularly relates to a wheat yield heterosis related gene TalncRNA1809, and a coding gene and application thereof.

Background

Long non-coding RNAs (lncrnas) are non-coding RNAs, typically greater than 200 nucleotides in length, containing 1 Open Reading Frame (ORF) of less than 100 amino acids and having no protein coding function. Long non-coding RNA is a key regulatory element involved in many growth and development processes and stress response in organisms. They share similarities with mRNA in terms of splicing, polyadenylation, and conserved sequences. Two plant-specific RNA polymerases evolved from RNA polymerase II (RNA Pol IV and Pol V) triggered the transcription of IncRNA. The polymerases regulate gene expression through gene silencing mechanisms and epigenetic regulation. The incrna can be polyadenylated or non-polyadenylated, which is generally shorter and less expressed than polyadenylated incrna. lncRNA can be transcribed from any location in the genome. Lncrnas can be classified into five types according to their relative positions and orientations in the genome with genes encoding similar proteins: a sense long non-coding RNA; antisense transcribed RNA derived from introns; intergenic non-coding RNA; non-coding RNA within a gene; bidirectional non-coding RNA.

So far, the function of lncRNA in plants is not only found in model plants such as arabidopsis thaliana and rice, but also reported in plants such as corn, wheat, millet, cotton, cabbage and cucumber. Compared with protein-encoding genes, lncrnas have tissue expression specificity in plants, participate in different biological metabolic pathways, and function through a variety of mechanisms, including activation, aggregation, or transport of proteins to produce epigenetic silencing and suppression, nucleosome-directed modification of promoter activity and regulation of DNA and histone methylation to produce epigenetic modifications.

The major biological functions of lncRNA are: participate in a variety of important regulatory processes, such as X chromosome silencing, genomic imprinting, chromatin modification, transcriptional activation, transcriptional interference, intranuclear transport, and the like; protein-encoding genes are protected in a variety of modes. Many lncrnas occur only at specific developmental stages or are tissue or cell specific, and many have conserved secondary structures and splicing patterns. The plant lncRNA plays an important biological function in the aspects of gene expression regulation, pollen development, lateral root development, male sterility, disease resistance, abiotic stress response and the like.

The lncRNA realizes the regulation of gene expression mainly from three aspects of epigenetics, transcriptional regulation and post-transcriptional regulation. In epigenetics, lncRNA can play a role by recruiting specific modification complexes to corresponding sites, so that the DNA methylation state is changed, and can also directly act with DNA methyltransferase to regulate and control the expression of corresponding genes. IncRNA itself is also affected by methylation modifications, which can cause different expression patterns and thus affect the expression of the target gene. In addition, lncRNA can promote different types of histone modification to influence the expression of target genes. At the transcriptional level, incRNA primarily affects mRNA production, and part of incRNA is located in the promoter region upstream of the coding gene and acts as a cis-acting element to interfere with transcription of downstream genes, thereby affecting mRNA production. At the post-transcriptional level, lncRNA can affect pre-mRNA splicing, intranuclear transport and mRNA degradation, and can also regulate gene expression in the form of double-stranded complexes with pre-mRNA.

lncRNA plays a role in regulation and control in plant growth and development. The FLOWERING LOCUS C (FLC) is found to participate in vernalization to induce FLOWERING in Arabidopsis; two Arabidopsis thaliana RNA-binding proteins (At NSRs) are involved in lateral root development regulation. After the expression of lncRNA-COLDAIR is inhibited by an RNA interference (RNAi) technology, the flowering time delay of an Arabidopsis plant is found, and the lncRNA plays a role in regulating and controlling the transcription and silencing of related genes in the flower development process. DRIR is a forward-regulated lncRNA of arabidopsis drought and salt stress responses, which may be involved in regulating plant responses to abiotic stress by modulating the expression of a series of genes involved in stress responses. The TPSI1(tomato phosphate stability induced 1) gene family is a relatively clear stress response lncRNA which is firstly found in tomato, a TPSI1 family member induces expression under phosphorus stress, and homologous genes of the TPSI1 family member are also detected in arabidopsis thaliana and rice. The lncRNA (Zm401) specifically expressed by the pollen in the corn can regulate the expression of 3 key genes related to the development of the corn pollen, and finally, male sterility is caused. LDMAR is lncRNA related to rice photosensitive male sterility. Two lncRNAs (bra-eTM160-1 and bra-eTM160-2) exist in the processes of cabbage pollen development and pollination fertilization, can be used as functional eTM to regulate the activity of cabbage miRNA160, and further influence the expression of target gene ARF family members to participate in pollen development. The ricinus lncRNA and the adjacent protein coding gene show strong coexpression, and the lncRNA is suggested to play an important role in regulating the development of embryo and endosperm.

Disclosure of Invention

The invention aims to provide a wheat yield heterosis related gene TalncRNA1809 and a coding gene thereof.

It is still another object of the present invention to provide a recombinant vector comprising the above gene.

Another object of the present invention is to provide a recombinant strain comprising the above gene.

Still another objective of the invention is to provide application of the gene TalncRNA1809 related to wheat yield and heterosis and a coding gene thereof.

It is a further object of the present invention to provide a method for increasing plant yield.

Wheat yield heterosis related gene TalncRNA1809 code gene according to the embodiment of the invention

Has a cDNA sequence shown as SEQ ID NO. 1:

CCATCGATGCCCAGGGCAGCGAGCTGGCTAGGTAGAGTCGAGACCGAGAGAAATGGAACA

TGGCATCATCTTTGTGATGCTCCTGGCCATGGCGGCCACCGTCGGAGCGCGGTCAGGAGC

CACGGCGCAGCCATCCTGCATCCCCACTGCAGATGGCGCCCCTCGCATGCACGCCGGAGC

AGTGCCCCCGTCGGTCGCCGCCAACTCGTCCCCGGCGCTTCATCGTCTGCATCGTCCTGC

CAGAGGACTGCCAACGCACCCAGCTCCGGCGGATCATGAACCGCGGTCACTCGCCCGGCC

GCTTCCTCGTCTGCCTCGTCCGGCCAGAGGACTACCGACAACACCAACTCCCGTGGATCA

TGAGCCGTGGTCACCACCGCCGCCCGCCATGCAAGGGTGATTAGTTTATGAAAGTGCAGC

CGTGCCTGTTTCTCAGTACGTTGCTGTTGATCACCTGGCAAAAAGAAAAGACCCAGTGTC

GTCGCTTTAATATTCTGTATCCTGTAGTGTAGTTCAGTCTAGTTCCAGTTTTTGTCAGCG

TGCAAGTTCTAGTCTACATTTTTTGCTTAAACATCACTGTAATAAGTTCTTTAGGCCTTA

CATCTAAATTCGCGTCTTCTCTTGGAATCTTTTTTCTTTGAACACCTCTTTGAATCTTT

the invention also provides an expression cassette, a recombinant expression vector, a transgenic cell line and a recombinant strain containing the TalncRNA1809 gene.

The plant expression vector according to the embodiment of the present invention includes binary agrobacterium vectors, vectors that can be used for plant microprojectile bombardment, and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal can lead polyadenylic acid to the 3 'end of the mRNA precursor, and the untranslated regions transcribed from the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (e.g., nopaline synthase Nos genes) and plant genes all have similar functions.

When the talncRNA1809 is used for constructing a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added in front of the transcription initiation nucleotide, such as a cauliflower mosaic virus (CaMV)35S promoter and a maize Ubiquitin promoter (Ubiquitin), and the enhanced promoter or constitutive promoter can be used independently or combined with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers having resistance (gentamicin marker, kanamycin marker, etc.), or chemical-resistant agent marker genes (e.g., herbicide-resistant gene), etc., which are expressed in plants. From the viewpoint of safety of the transgenic plant, any selectable marker gene may not be added.

A method for increasing plant yield according to an embodiment of the present invention comprises the step of overexpressing in a plant yield heterosis-related gene TalncRNA 1809. Any vector capable of guiding the expression of the exogenous gene in the plant is utilized to introduce the TalncRNA1809 gene into plant cells, so that a transgenic cell line and a transgenic plant with enhanced yield advantage can be obtained. The expression vector carrying the encoding gene can be used to transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium mediation, etc., and the transformed plant tissues can be cultivated into plants. The plant host to be transformed may be either a monocotyledonous or dicotyledonous plant, such as: rice, corn, cucumber, tomato, poplar, lawn grass, alfalfa and the like.

The invention has the beneficial effects that:

according to the invention, 179 new species of the national examined hybrid wheat with strong yield heterosis are taken as experimental materials, the TalncRNA1809 related to yield heterosis and the coding gene thereof are obtained and are introduced into wheat, and the yield heterosis of plants is obviously improved. The lnRNA related to the yield heterosis has very important theoretical and practical significance for improving and enhancing the wheat yield heterosis, increasing the yield and accelerating the high-yield molecular breeding process.

Drawings

Fig. 1 shows the cDNA cloning results of the wheat yield heterosis-related TalncRNA1809 gene, wherein M: trans2K Plus DNAmarker; 1 is a cDNA amplification product of a TalncRNA1809 gene;

FIG. 2 shows the results of analysis of the expression patterns of the TalncRNA1809 gene in different strong, medium and weak hybrid combinations, P1 is different sterile line materials; p2 is a different restorer material; f1 is a different hybrid material.

FIG. 3 shows a subcellular localization analysis of TalncRNA1809, in which 35S: GFP is empty vector, 35S: TalncRNA1809-GFP is fusion vector, Bright is Bright field channel, GFP is green fluorescent protein channel, Merged is the superposition of two channels GFP and Bright, (ruler length: 10 μm);

fig. 4 shows the detection of the TalncRNA1809 transgenic wheat molecule and the yield analysis, wherein, M: trans2K PlusDNAmarker; 1 is a positive control; 2-7 are different transgenic strains of the TalncRNA1809 gene; WT is wild type wheat Fieldier; in the Ubi, the TalncRNA1809 is an overexpression transgenic wheat strain.

Detailed Description

The molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.