Poplar leaf type development control gene and application thereof

A poplar, gene technology, applied in the field of plant genetic engineering and biology

Inactive Publication Date: 2019-04-26
INST OF FORESTRY CHINESE ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, in woody plants, there are few reports on the cultivation of new varieties of leaf type development

Method used

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  • Poplar leaf type development control gene and application thereof
  • Poplar leaf type development control gene and application thereof
  • Poplar leaf type development control gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Clone PagKNAT2 / 6b Gene

[0049] Using 84K (P.alba X P.glandulosa) Populus albadensis as material, use RNeasy Plant Mini kit and RNase-free DNase I kit (Qiagen, Hilden, Germany) to extract the total RNA of one-month soil culture seedlings, each sample About 1.0 μg of RNA was taken, and the first strand of cDNA was synthesized by using the SuperScript III first-strand synthesis system (LifeTechnologies, Carlsbad, CA, USA). Referring to the published Populus trichocarpa genome sequence, primers were designed using Primer 5 software (the amplicon included start codon and stop codon) for full-length gene amplification (GATEWAY linker is introduced into the primer);

[0050] Wherein, the PagKNAT2 / 6b ORF forward primer is shown as sequence 3 in the sequence listing, see Table 1 below;

[0051] Table 1

[0052]

[0053] The sequence of the PagKNAT2 / 6b ORF reverse primer is shown as sequence 4 in the sequence listing, see Table 2 below;

[0054] Table 2

[0055]...

Embodiment 2

[0063] Embodiment 2 Construction of PagKNAT2 / 6b gene plant expression vector

[0064] Using cloning technology to construct an overexpression vector of the PagKNAT2 / 6b gene, using specific PCR primers (the PagKNAT2 / 6b ORF primer in Example 1), and using 84K cDNA as a template, PCR amplification was carried out, and the PagKNAT2 / 6b gene ORF was constructed into the entry vector , the entry vector is PDNOR207, and the sequence is shown as sequence 5 in the sequence listing, see Table 5 below;

[0065] table 5

[0066]

[0067]

[0068]

[0069] The reaction system is Fresh PCR product (fresh PCR product) 150ng; PDNOR207 vector (vector) 75ng; BP Clonase (cloning enzyme) II enzyme mix (mixed enzyme) 0.8μl; sterile ddH 2 Make up to 4 μl with O; the reaction procedure is: react at 25°C for more than 5h.

[0070] Pick positive clones from the screening culture plate for PCR detection and sequencing verification. After the entry vector with PagKNAT2 / 6b gene is linearized by...

Embodiment 3

[0080] The genetic transformation of embodiment 3PagKNAT2 / 6b gene

[0081] By electric shock method, the PMDC32-PagKNAT2 / 6b overexpression vector constructed is transferred in the Agrobacterium GV3101, and by Agrobacterium-mediated, the PagKNAT2 / 6b gene is transferred into poplar (Yinden poplar, 84K poplar, the same below), The transformation steps are as follows: the hybrid poplar clone 84K tissue culture seedlings used for genetic transformation were cultivated at a temperature of 23-25°C, a light of 16 / 8h (day / night), and a light intensity of 50 μM m -2 the s -1 Cultured under the condition of , the Agrobacterium containing the PMDC32-PagKNAT2 / 6b expression vector infects 84K leaf discs at OD600=0.6~0.8, and the infected leaf discs are grown in adventitious bud induction medium (SIM, Murashige-Skoog (MS) Add 0.5mg / l 6-benzyl aminopurine (6-BA, 6-benzyl aminopurine) and 0.05mg / l naphthalene acetic acid (NAA, naphthaleneacetic acid)) to the minimal medium, in dark conditions...

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Abstract

The invention relates to a poplar leaf type developmental control gene and application thereof, which belong to the technical field of plant genetic engineering and the biological field. A nucleotidesequence of the poplar leaf type development control gene PagKNAT2 / 6b is as shown in a sequence 1 in a sequence table; an amino acid sequence, expressing protein, of PagKNAT2 / 6b is as shown in sequence 2 in the sequence table. The PagKNAT2 / 6b gene is shifted into 84k poplar, and the transgenic polar which excessively expresses PagKNAT2 / 6b is compared with a wild type, so that an obvious rolled andfolded leaf type appears, and therefore, the PagKNAT2 / 6b gene is the key of controlling poplar leaf development, and has an important application value in the woods genetic engineering field and theclonal forestry field.

Description

technical field [0001] The invention relates to a transcription factor for regulating the development of poplar leaves and its application, in particular to a gene for regulating the development of poplar leaf shape and its application, belonging to the fields of plant genetic engineering and biotechnology. Background technique [0002] As an economic tree species widely distributed in the world, poplar has become the most famous fast-growing timber forest and greening tree species in my country by virtue of its rapid growth, easy reproduction, wide wood use and strong adaptability. Its cultivation and breeding work has always been valued . [0003] In recent years, with the completion of poplar genome sequencing, molecular biology techniques have been gradually applied to the genetic improvement of poplar. Poplar has many varieties, luxuriant branches and leaves, and leaves fall late in autumn, making it one of the important street trees. Ornamental characteristics such as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/10C12N15/82A01H5/00A01H6/00
CPCC07K14/415C12N15/1096C12N15/8241C12Q2531/113
Inventor 卢孟柱赵岩秋宋学勤
Owner INST OF FORESTRY CHINESE ACAD OF FORESTRY
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