Application of lotus metallothionein gene MT2a in improving seed longevity and activity

A technology of metallothionein and gene, applied in the field of molecular biology and biology, can solve problems such as the mechanism is not very clear, achieve the effects of improving storage life and vitality, important economic benefits and application prospects, and reducing agricultural losses

Inactive Publication Date: 2011-08-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although extensive research has been done on seed lifespan a

Method used

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  • Application of lotus metallothionein gene MT2a in improving seed longevity and activity
  • Application of lotus metallothionein gene MT2a in improving seed longevity and activity
  • Application of lotus metallothionein gene MT2a in improving seed longevity and activity

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: lotus metallothionein gene MT2a clone

[0033] (1) Preparation of hypocotyls of lotus seeds: Harvest the lotus seeds at the late stage of development and strip off the hypocotyls;

[0034](2) Extraction of total RNA: Total RNA was extracted using Trizol product from Invitrogen;

[0035] (3) cDNA library construction: the library was constructed using SMART cDNA library construction kit from Clontech;

[0036] (4) EST sequencing: The obtained positive monoclonal bacterial liquid is used as a sequencing sample, and Shanghai Boya Biotechnology Co., Ltd. is entrusted to use a 3730 sequencer to sequence the positive monoclonal after PCR verification;

[0037] (5) Homologous search: use BLAST tool to search for the homologous gene of the obtained EST sequence in GenBank, and confirm that the obtained gene is the lotus metallothionein gene.

Embodiment 2

[0038] Embodiment 2: Construction of plant expression vector

[0039] (1) Cloning of gene fragments: 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 enzyme cutting site.

[0040] Forward primer: SEQ ID NO: 3:

[0041] 5′-TCC CCCGGG AATGTCTTGCTGCGGAGGA-3′

[0042] Reverse primer: SEQ ID NO: 4:

[0043] 5′-CTG GAGCTC GCCCTC CTCTCATTTGCAG-3′

[0044] 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°...

Embodiment 3

[0050] Example 3: Genetic Transformation of Arabidopsis

[0051] 1. Pre-treatment of Arabidopsis transformation

[0052] 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.

[0053] Two preparation of dipping medium

[0054] 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, and the operation needs to be gentle.

[0055] 3 Agrobact...

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Abstract

The invention belongs to the fields of molecular biology and biotechnology, and particularly relates to an application of a lotus metallothionein gene MT2a in improving plant seed longevity and activity. A lotus seed maturation later-stage embryo axis cDNA (complementary deoxyribonucleic acid) library is constructed, EST sequencing is performed to obtain a full-length lotus metallothionein gene which is named as MT2a, and the accession number of the full-length lotus metallothionein gene in GenBank is EF421200. A sense over-expression vector of the MT2a is further constructed, the model plantarabidopsis thaliana is transformed, and the result indicates that the transgenic arabidopsis thaliana seed has stronger capability in resisting artificial aging and germination activity under sodiumchloride stress than the wild seed. Therefore, if the MT2a is transferred into important crops such as rice, wheat, corn, soybean and the like, the storage life and activity of the crop seeds can be possibly prolonged. Due to the application of MT2a, the global tremendous agricultural loss caused by seed deterioration and salt stress every year can be reduced, and the MT2a has important economic benefits and application prospects.

Description

technical field [0001] The invention belongs to the field of molecular biology and biotechnology, and specifically relates to the lotus metallothionein gene MT2a Application in improving the longevity and vigor of plant seeds. Background technique [0002] Seeds are the foundation of agricultural production. Seed longevity and vigor play a decisive role in plant reproduction, crop yield, germplasm conservation and biodiversity. After long-term storage or under adverse conditions, high-quality seeds can still maintain a high germination rate and grow robust seedlings. However, with the prolongation of storage time, most of the seeds began to deteriorate, gradually lost their vigor, became more and more sensitive to adversity during germination, and finally failed to germinate. Seed deterioration is usually accompanied by a series of physiological and biochemical changes, such as DNA breakage, lipid peroxidation, and protein damage. McDonald et al. believed that the lifesp...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415A01H5/00
Inventor 黄上志周玉亮黎茵楚璞陈虎辉
Owner SUN YAT SEN UNIV
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