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Lotus annexin and expression vector and application thereof

A technology for expressing vectors and annexins, applied in the fields of molecular biology and plant genetic engineering, can solve the problems of no annexins and heat resistance of seeds, achieve broad application space and market prospects, improve heat resistance and Vitality, the effect of improving germination vitality

Inactive Publication Date: 2011-11-02
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Rhee et al. (Rhee, H.J., et al., 2000, Annexin I is a stress protein induced by heat, oxidative stress and a sulfhydryl-reactive agent. Eur J Biochem 267: 3220-3225) found that high temperature increased when animal cells were heat treated The expression of annexin I was detected, indicating that it may be a heat stress response protein, but there is no report on the relationship between annexin and seed heat tolerance in plants

Method used

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  • Lotus annexin and expression vector and application thereof
  • Lotus annexin and expression vector and application thereof
  • Lotus annexin and expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Lotus annexin gene NnANN1 clone

[0032] (1) Preparation of hypocotyls of lotus seeds: Harvest the lotus seeds at the late stage of development and strip off the hypocotyls;

[0033] (2) Extraction of total RNA: Total RNA was extracted using Trizol product from Invitrogen;

[0034] (3) cDNA library construction: the library was constructed using SMART cDNA library construction kit from Clontech;

[0035] (4) EST sequencing: The obtained positive monoclonal bacterial liquid is used as a sequencing sample, and Shanghai Boya Biotechnology Co., Ltd. is entrusted to sequence the positive monoclonal after PCR verification with a 3730 sequencer;

[0036] (5) Homologous search: BLAST tool was used to search the homologous gene of the obtained EST sequence in GenBank, and the obtained gene was confirmed to be the lotus annexin gene.

Embodiment 2

[0037] Example 2 Construction of Plant Expression Vectors

[0038] (1) Gene fragment cloning: Using the lotus cDNA library described in Example 1 as a template, PCR cloning was performed to obtain enzyme cleavage sites Sma I and Sac PCR fragment of I. The PCR primers are as follows, the underlined part is the restriction site:

[0039] Forward primer: SEQ ID NO:3: 5′-TCC CCCGGG ATGGCTACCATCACAGTCCCTG-3';

[0040] Reverse primer: SEQ ID NO:4: 5'-CTG GAGCTC TCACAGCTCTTCGCACCCCAGT-3';

[0041] The PCR reaction system is: 2 μl lotus cDNA library, 6 μl dNTP (2.5 mM), 1.5 μl forward primer (10 μM), 1.5 μl reverse primer (10 μM), 5 μl 10×PCR buffer, 1 μl EX Taq enzyme (product of Takara Company), and deionized water was finally added to make the total system 50 μl. The reaction conditions of PCR are: 94°C for 3 minutes; then enter the following cycle: 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds, a total of 30 cycles; finally 72°C for 7 minutes.

[004...

Embodiment 3

[0047] Example 3 Genetic Transformation of Arabidopsis

[0048] 1. Pre-treatment of Arabidopsis transformation

[0049] When the main moss grows to 5-6cm, cut off the entire inflorescence at the base of the inflorescence, and remove the top advantage. After 1 week, 4-6 new side mosses will grow on the axillary buds, and the side mosses will form flower buds and partially bloom. Or when 1-2 siliques are formed, they can be used for transformation, and the grown siliques need to be cut off before transformation. The day before transformation, the plants were well watered and covered with a plastic bag to maintain a high humidity environment.

[0050] 2. Preparation of Infection Medium

[0051] The composition of the dipping medium used for soaking Arabidopsis flocs is 1 / 2MS medium containing 5% sucrose, pH=5.8 (adjusted with KOH), autoclaved. When using, add 0.02%-0.05% surfactant Silwet L-77 or surfactant Tween-20 (tween-20), and the operation needs to be gentle.

[0052]...

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Abstract

The invention discloses a lotus annexin and an expression vector and application thereof, and belongs to the technical field of plant gene engineering. The lotus annexin has an amino acid sequence shown as SEQ ID NO.1, and the nucleotide sequence of the coded gene of the lotus annexin is shown as SEQ ID NO.2. The invention also discloses the expression vector of the lotus annexin. The expression vector is formed by inserting the coded gene of the lotus annexin into a prokaryotic or eukaryotic expression vector. The lotus annexin can effectively improve the heat resistance of plant seeds and enhance the germinating activity of the seeds, and has wide application prospect in the fields of plant variety breeding and agricultural production.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and plant genetic engineering, in particular to a lotus annexin for improving heat resistance and seed vigor of plant seeds and an application thereof. Background technique [0002] High temperature is one of the important environmental factors affecting plant growth, development and reproduction. Due to the influence of the greenhouse effect, the trend of global warming is becoming more and more obvious, and the damage caused by the decrease of seed vigor and survival rate caused by high temperature to crop quality and yield is also increasing. The key gene of vitality has important scientific significance and economic value. [0003] lotus( Nelumbo nucifera Gaertn.) seeds are one of the longest-lived and most heat-resistant seeds so far confirmed (; Shen-Miller, et al., 1995, Exceptional seed longevity and robust growth: ancient sacred lotus from China. Am J Bot 82: 1367-1380;), i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N15/66C12N1/21A01H5/10C12R1/01
CPCY02A40/138
Inventor 黄上志楚璞陈虎辉黎茵周玉亮
Owner SUN YAT SEN UNIV
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