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Key gene LhHB9 for somatic embryo radicle elongation of liriodendron hybrids and application thereof

A technology of Liriodendron body and key genes, which is applied to the key gene LhHB9 of hybrid Liriodendron radicle elongation and its application field, can solve the problems of inconsistent development, small number, small individuals, etc., and achieve good practicability Effect

Active Publication Date: 2019-06-25
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because zygotic embryos are deeply buried in the embryo sac, and have the characteristics of small individuals, small numbers, and inconsistent development, it is difficult to conduct in-depth and detailed research on early zygotic embryos.

Method used

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  • Key gene LhHB9 for somatic embryo radicle elongation of liriodendron hybrids and application thereof
  • Key gene LhHB9 for somatic embryo radicle elongation of liriodendron hybrids and application thereof
  • Key gene LhHB9 for somatic embryo radicle elongation of liriodendron hybrids and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1 LhHB9 gene cloning

[0020] 1. Extraction of hybrid tulip tree RNA

[0021] The leaves of Liriodendron tulipifera were used as materials to extract RNA. The specific steps are: (1) Use liquid nitrogen to fully pre-cool the mortar until the liquid nitrogen is added to the mortar until it no longer boils; (2) Put no more than 50mg of plant tissue into Fully grind in a mortar filled with liquid nitrogen (2-3 times); (3) When the liquid nitrogen is partially volatilized and the sample is not melted, add 1mL of lysate, continue to grind until the sample is completely mixed with the lyse, and place at room temperature (4) Use a pipette gun to transfer the homogenate to a 2mL centrifuge tube; (5) Centrifuge at 12000r / min for 2 minutes at room temperature, absorb the upper layer liquid and transfer it to a new tube; (6) Add an equal volume of 24:1, vortex and mix well, centrifuge at 12000r / min for 2 minutes, absorb the uppermost liquid and transfer to a new tube, ...

Embodiment 2

[0041] Embodiment 2 LhHB9 gene functional verification

[0042] 1. Construction of LhHB9 overexpression vector

[0043] The basic vector used in the construction of the target gene and target gene mutant transient expression vector is PJIT166. The vector was constructed using the method of homologous recombination. The reagent used was Gibson of NEB Master mix, the entire assembly process can be found on the NEB website https: / / www.neb.com / applications / cloning-and-synthetic-biology / dna-assembly-and-cloning / g ibson-assembly.

[0044] (1) PCR to generate insert fragments

[0045] Use the NEBuilder online website (http: / / nebuilder.neb.com / ) to design primers, and the primer sequences are as follows:

[0046] PJIT166-LhHB9F: 5′-ggagaggacagcccaagcttATGGCGCTTGCGATGCAC-3′,

[0047] PJIT166-LhHB9R: 5'-gctcaccatggatcctctagaAAGAAAAGACCAGTTCATGAACAAGAAGGC-3'.

[0048] Target gene and T-Vector pMD TM 19 (Simple) The positive plasmid with correct sequencing was used as a template, ...

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Abstract

The invention discloses a key gene LhHB9 for somatic embryo radicle elongation of liriodendron hybrids and application thereof. The DNAsequence of the key gene LhHB9 for somatic embryo radicle elongation of liriodendron hybrids is as shown in SEQ ID NO. 1. A cDNA sequence of the LhHB9 gene of liriodendron hybrids is cloned, an overexpression vector of the LhHB9 gene is constructed, liriodendron hybrids embryogenic callus is taken as a receptor material, and the genetic transformation is carried out by an agrobacterium-mediated method. After transgenic callus is obtained by G418 screening, it is detected and the transgene was preliminarily determined to be sucessful. Through the induction of the LhHB9 gene transgenic callus embryo, it is found that the transgenic somatic embryo radicle hasobvious elongation. The statistical data shows that the 35S:LhHB9 somatic embryo radicle is the most significant compared with the control CK, indicating that overexpression of the target gene can promote the elongation of the radicle and has good practicability.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a key gene LhHB9 for the elongation of the radicle root of a hybrid tulip tree and its application. Background technique [0002] Plant somatic embryogenesis (referred to as "somatic embryogenesis") refers to the process of somatic cells producing new individuals under in vitro conditions. Steward was the first to obtain somatic embryos using carrot suspensions in 1958. Since then, multiple species have obtained whole plants through the somatic embryo regeneration pathway. Somatic embryogenesis is a common phenomenon in plants, and most of them can induce somatic embryo regeneration by using different organs and tissues and suitable external conditions. Because zygotic embryos are deeply buried in the embryo sac, and have the characteristics of small individuals, few numbers, and inconsistent development, it is difficult to conduct in-depth and detailed ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/00
Inventor 陈金慧陆凤娟郝兆东陈桢雨陈新颖翁禹豪施季森成铁龙汪贵斌
Owner NANJING FORESTRY UNIV
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