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Peanut senescence gene for regulating programmed cell death, coding sequence and application

A technology of programmed death and coding sequence, which is applied in the field of peanut aging gene AhSAG and its coding sequence and application, to achieve the effect of maintaining adaptability

Inactive Publication Date: 2012-09-12
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Peanut aging gene AhSAG has not been published so far

Method used

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  • Peanut senescence gene for regulating programmed cell death, coding sequence and application
  • Peanut senescence gene for regulating programmed cell death, coding sequence and application
  • Peanut senescence gene for regulating programmed cell death, coding sequence and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1: RACE method obtains the full-length gene of AhSAG

[0071] 1. Synthesis of cDNA

[0072] According to different requirements, reverse transcription was performed to obtain different cDNAs, as shown in Table 3.

[0073] Synthesis of table 3AhSAG middle fragment, 3' end and 5' end cDNA

[0074] template

reverse transcription primer

purpose

99-1507400μmol / L Al treatment for 4d

OligoT18

normal template

99-1507400μmol / L Al treatment for 4d

F

3'race template

99-1507400μmol / L Al treatment for 4d

SAG 5′ reverse transcription

5'race template

[0075] 2. PCR amplification of the middle fragment, 3' end and 5' end cDNA fragment (50 μL system)

[0076] (1) Middle segment

[0077] reaction system:

[0078] The operation was performed on ice, and the specific operating conditions are shown in Table 3.

[0079] Table 3: Reaction system operating conditions

[0080] response system

v...

Embodiment 2

[0168] 实施例2、AhSAG cDNA正、反义植物表达载体的构建

[0169] 利用实施准备的材料和实施例1得到的AhSAG基因,将AhSAG基因插入载体pBI121-GFP(含GFP荧光蛋白)中GUS基因和35S启动子之间,并跟NOS终止子构成一个完整的表达框架。利用正义引物和反义引物通过PCR扩增基因AhSAG的开放阅读框(ORF),纯化回收后,克隆到空载体pTG19-T(上海生工),获得正义质粒pTG19-AhSAG和反义质粒pTG19-anti-AhSAG。将含有AhSAG的pTG19-AhSAG和pTG19-anti-AhSAG cDNA的克隆载体用BamH I和XbaI双酶切,分别回收小片段,定向连接到经BamH I和XbaI双酶切的载体pBI121-GFP上,连接产物转化E.coli DH5α感受态细胞、碱法提取质粒。正义载体命名为pBI121-GFP-AhSAG;反义载体命名为pBI121-GFP-anti-AhSAG。

[0170] 1、AhSAG基因编码区cDNA序列的克隆

[0171] (1)RT-PCR(50μL体系)

[0172] reaction system:

[0173] 采用冰上操作,具体操作条件见表13。

[0174] 表13:反应体系操作条件

[0175] response system

volume

10×Buffer (including Mg 2+ )

5μL

dNTP (2.5mM)

4μL

P1: SAG sense / antisense upstream (10 μM)

2μL

P2: downstream of SAG sense / antisense (10 μM)

2μL

OligoT18 reverse transcription product stock solution

1μL

Ex-Taq

0.25 μL

wxya 2 o

35.75μL

total

50μL

[0176] Reaction conditions:

...

Embodiment 3

[0206] 实施例3:AhSAG基因在不同品种和不同铝胁迫下表达量的变化

[0207] 选用Actin基因做内参,提取8个材料的RNA,以oligoT18为引物进行反转录,得到8个材料的cDNA,分别记为99-0、99-20、99-100、99-400、中2-0、中2-20、中2-100和中2-400,进行RT-PCR(Real-time PCR)实时。

[0208] (1)反应体系:

[0209] 采用冰上操作,具体操作条件见表17。

[0210] 表17:反应体系操作条件

[0211] reaction system

volume

Hotstart Fluo-PCR Mix

12.5μL

P1 (10μM)

1μL

P2 (10μM)

1μL

cDNA (100mM)

0.5μL

wxya 2 o

10μL

total

25 μL

[0212] (2)反应条件:

[0213] 具体反应操作条件见表18。

[0214] 表18:反应操作条件

[0215]

[0216]

[0217] 根据2 -△△t 法计算相对表达量。

[0218] 结果:中花2号和99-1507的表达量的趋势基本一致,只是在低浓度下,中花2号的表达量有所下降,有可能是低浓度的Al促进了植物的生长,导致衰老基因的表达下调。但当在中、高浓度处理时,衰老基因的高丰度表达。由此,可以认为AhSAG基因属于衰老诱导增强型基因,中、高浓度下诱导AhSAG等衰老基因超表达,促进植物早衰。

[0219] From the expression of AhSAG gene and the relative expression of AhSAG gene between Zhonghua 2 and 99-1507, the expression of the aluminum-sensitive variety Zhonghua 2 suddenly increased under the treatment of medium and hi...

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Abstract

The invention discloses a peanut senescence gene for regulating programmed cell death, a coding sequence and application. RNA (ribonucleic acid) is extracted from root tip of peanut, and is cloned to a peanut senescence gene AhSAG, and the ORF (open read frame) of the peanut senescence gene is a cDNA (complementary deoxyribonucleic acid) sequence with coding sequence of 474bp and codes of 157aa; the AhSAG comes from a peanut root tip meristematic tissue of aluminum-induced peanut root tip generating programmed cell death, and is a senescence induction enhanced gene to induce, activate and promote progeria of plants. The peanut senescence disclosed by the invention can be expressed in tobacco, and can be used for regulating the adaptability of plant cells to environment changes and maintaining environmental stability and synthesis capacity in the cell.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to the peanut senescence gene AhSAG for regulating programmed cell death, its coding sequence and application. Background technique [0002] In plants, cell death is a common phenomenon and is part of a highly specialized cellular development process. During this process, plant cells are stimulated by extrinsic and intrinsic information (external environment, developmental signals, metabolic environment, etc.), which change cell fate and cause cell death. There are two types of cell death: cell necrosis and programmed cell death (PCD). Cell necrosis is usually due to the release of lysosomal granules caused by accidental external factors such as extreme stimulation such as heat or drugs, resulting in cell autolysis and death, which is an abnormal death. PCD refers to a series of ordered molecular events regulated by genetically encoded instructions, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/84C12N1/21A01H5/00
Inventor 詹洁何龙飞何虎翼王天菊
Owner GUANGXI UNIV
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