阅读说明：本技术 一种提高杨树木材产量的方法 (Method for improving wood yield of poplar ) 是由 王殿 周功克 柴国华 孔英珍 贺郭 王丛鹏 于 2021-07-22 设计创作，主要内容包括：本发明专利涉及分子生物学、基因工程技术等领域,具体涉及从杨树南林895品种中鉴定出一段具有调控功能的DNA序列,对其功能和应用进行了研究。其cDNA序列如序列表1所示。本发明还公开了利用该片段促进杨树生长和调控杨树产量的方法。(The invention relates to the fields of molecular biology, genetic engineering technology and the like, in particular to a DNA sequence with a regulation function identified from a poplar nanlin 895 variety, and researches on the function and application of the DNA sequence. The cDNA sequence is shown in a sequence table 1. The invention also discloses a method for promoting the growth of poplar and regulating the yield of poplar by using the fragment.)

1. Poplar treePdCPD1.2（Potri.010G189800.2) The sequence of the 3' UTR segment of the gene is shown as the nucleotide sequence in the sequence table 1.

2. A gene silencing vector comprising the nucleotide sequence of claim 1.

3. Use of the gene silencing vector according to claim 2, wherein the vector according to claim 2 is transformed into a crop of poplar, rice, wheat, cotton or maize to promote plant growth and increase yield.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to application of a gene silencing vector for transforming a 3' UTR fragment sequence of a gene of PdCPD1.2 (Potri.010G189800.2) of a poplar in improving plant yield.

Background

The poplar is one of the most main artificial forest tree species in temperate regions in the world, has the advantages of high growth speed, strong adaptability, early yield, large wood accumulation and the like, is rich in genetic background, is easy to carry out rapid asexual propagation and genetic transformation, and can become an ideal woody pattern plant along with the completion of whole genome sequencing of Chinese white poplar and populus euphratica. Therefore, the method for improving the yield of the poplar by taking the poplar as a research object has important significance for improving the wood yield.

Brassinolide (BRs) belongs to the group of steroid hormones, one of the six major hormones. BR can be identified by a receptor BRI1 on a plant cell membrane, signals are transmitted to two homologous central control hubs BZR1 and BES1 through a series of phosphorylation and dephosphorylation processes, and thousands of downstream target genes are activated or inhibited at different stages of different tissues to accurately control the growth and adversity stress processes such as cell elongation, xylem development, apical dominance, organ senescence, adversity stress and the like. Currently, some studies of BR regulation of xylem development have been reported in arabidopsis thaliana and rice. The arabidopsis and rice mutants are identified, and the phenomenon that BR synthesis and signal transduction are blocked causes the plants to be short and the xylem of the stems is reduced is found. Furthermore, arabidopsis BZR1 and BES1 redundantly regulate xylem and phloem cell differentiation. The arabidopsis stems were examined carefully and BRs were found to accumulate mainly in the cambium and developing xylem. These results indicate that BR affects herbaceous plant xylem cell differentiation.

CPD/CYP90A1 is cytochrome mono-oxidase, catalyzes the oxidation reaction of C-3 position of intermediate product in BRs synthesis containing 3 beta hydroxyl or C-22 hydroxyl and C22 and C23 dihydroxyl, and plays an important role in the process of converting compound 6-deoxyCT into 6-deoxyTE and converting CT into TE. CPD has different catalytic efficiency on different substrates, and the catalytic efficiency on substrates containing a delta 5-double bond and C-22 hydroxyl side chain is obviously higher than that of substrates containing C-22 and C-23 dihydroxyl side chains, and DET2 is found to have higher affinity on substrates containing monohydroxy or dihydroxyl side chains, which suggests that the CPD-mediated early C-22 hydroxylation pathway is the main pathway for BRs synthesis. In terms of biological function, two different insertion site mutants of the arabidopsis CPD gene, CPD and cbb3, both exhibit the typical characteristics of BR deletion: cell elongation is hindered, hypocotyl becomes short and tip back hooking is absent under dark culture. These characteristics are more severe than the mutant DET2 of the BR synthase DET2, and the addition of CPD gene mutation can prevent DET2 substrate accumulation, indicating that CPD is superior to DET 2. Further research shows that the CPD gene can be feedback-regulated by BR signals. When BR signals are activated, BR-specific transcription factors BZR1 and BES1 dephosphorylate, thereby directly inhibiting CPD expression at the transcriptional level, reducing BR synthesis. Therefore, the modification of the CPD gene can change the synthesis of BRs of the plant and further influence the growth of the plant.

Disclosure of Invention

The invention aims to provide a method for improving the yield of poplar.

In order to achieve the purpose, the invention adopts the technical scheme that:

in the first aspect of the invention, a 3' UTR segment of PdCPD1.2 gene of poplar is provided, and the sequence of the segment is shown in a sequence table 1.

In a second aspect of the invention, a gene silencing vector containing a 3' UTR segment of a PdCPD1.2 gene of a poplar is provided, and the vector comprises a microorganism expression vector and a plant expression vector.

In a third aspect of the invention, an application of a 3' UTR fragment of a PdCPD1.2 gene of a poplar in improving the yield of the poplar is provided.

The invention discovers that a CPD encoding gene PdCPD1.2 (Potri.010G189800.2) is abundantly expressed in Developing xylem (Developing xylem) from poplar transcriptome data, and means that the gene is possibly involved in wood formation. The RNAi carrier is constructed to inhibit the 3' UTR of the PdCPD1.2 gene, so that the expression of PdCPD1 can be properly activated, and the growth of poplar trees is promoted. The invention thus contributes to increasing the biomass of the forest.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention, are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 shows that the yield of poplar is improved after the 3' UTR segment of PdCPD1.2 gene is silenced.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, elements, and/or combinations thereof, unless the context clearly indicates otherwise.

In order to promote the research and application of genetic engineering in improving the yield of forest trees, the invention provides a 3' UTR fragment of PdCPD1.2 gene of poplar, the sequence of which is shown in a sequence table 1.

In a preferred technical scheme of the invention, the amplification of the 3' UTR segment of the PdCPD1.2 gene of the poplar comprises the following steps:

(1) taking a 3' UTR fragment of the PdCPD1.2 gene of the poplar as a template, and designing a primer;

(2) extracting total RNA of poplar seedlings, and then carrying out reverse transcription to obtain cDNA;

(3) and (3) taking the cDNA obtained in the step (2) as a template, performing PCR amplification by using high-fidelity enzyme, and recovering a purified product to obtain the cDNA.

In a second aspect of the invention, a gene silencing vector containing a 3' UTR segment of a PdCPD1.2 gene of a poplar is provided, and the vector comprises a microorganism expression vector and a plant expression vector.

In a third aspect of the invention, an application of a 3' UTR fragment of a PdCPD1.2 gene of a poplar in improving the yield of the poplar is provided.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1 cloning of PdCPD1.2 gene 3' UTR fragment of poplar and construction of RNAi vector

(1) Downloading the genome sequence of the PdCPD1.2 gene of the poplar from a phytozome website, analyzing a specific section of the genome sequence, and designing a primer to amplify the genome sequence;

(2) extracting the total RNA of the poplar seedling by using an Edley RNA extraction kit, and then carrying out reverse transcription on the RNA into cDNA by using a full-type gold reverse transcription kit;

(3) and (3) carrying out PCR amplification by using cDNA as a template and high-fidelity enzyme, recovering a purified product, constructing the purified product on an RNAi vector pUCC for sequencing, wherein the sequencing result is shown in a sequence table 1.

Sequence listing

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