Application of PLD (phospholipase D) epsilon gene in increasing nitrogen nutrition effective utilization and seed yield of crops

A nitrogen nutrition and gene technology, applied in the field of plant molecular breeding and biology, to achieve the effect of improving efficient reuse

Inactive Publication Date: 2014-05-07
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under the condition of N nutrient deficiency, the root growth of the PLDε deletion mutant was hindered, while the overexpression of PLDε promoted root growth, and the overexpression of PLDε showed that the growth of lateral roots and the number of lateral roots could be promoted under different nitrogen concentrations. increase, but it can only promote the growth of taproots in the absence of nitrogen nutrients

Method used

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  • Application of PLD (phospholipase D) epsilon gene in increasing nitrogen nutrition effective utilization and seed yield of crops
  • Application of PLD (phospholipase D) epsilon gene in increasing nitrogen nutrition effective utilization and seed yield of crops
  • Application of PLD (phospholipase D) epsilon gene in increasing nitrogen nutrition effective utilization and seed yield of crops

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: Rapeseed genetic transformation (dark light cultivation method)

[0033] Seed sterilization and germination: After soaking the seeds with 75% alcohol for 1 min, rinse the seeds with sterile water. Then use 0.15% HgCl 2 (containing 0.1% (v / v) Tween20) for 15 minutes, then rinsed with sterile water for 3-5 times; then sow the seeds in M 0 In the culture medium, 18 seeds were evenly sown per dish (3 cm in diameter). Put the petri dish into a sterile plastic culture box, and culture it at 25°C for 5 days under dark and light conditions.

[0034] Cultivation of Agrobacterium: Pick a single colony of Agrobacterium (GV3101, a strain containing the AtPLDα1 gene plasmid, purchased through a commercial channel) and put it in LB liquid culture medium (containing 50mg / L Km) for 1-2 days, the best The infection OD value is 0.6-0.8. Use a spectrophotometer to detect the concentration of the bacterial solution under the condition of 600nm light absorption. When the O...

Embodiment 2

[0044] Example 2: Identification and expression of PLDε transgenic plants

[0045] (1) Identification of PLDε transgenic plants

[0046] Transformed plants infected with Agrobacterium in M 4 After rooting was induced in the culture medium, an appropriate amount of leaf was taken to extract DNA, and the leaf DNA was detected by PCR with primer 35S-p / PLDε-A. The AtPLDε gene was successfully transferred. The electrophoresis results of the PCR products of some transformed plants showed that most of the plants could amplify fragments of the same size as the PCR products of the plasmid as a template, but only 2 transformed plants and WT DNA PCR products did not have target fragments, indicating that Agrobacterium The GV3101-mediated PLDε plasmid has been successfully transformed into rapeseed.

[0047] In the above method, the nucleotide sequences of the primer pairs used are as follows:

[0048] Primer pair 35S-p / PLDε-A:

[0049] Forward primer 35S-p5'-GAAGACGTTCCAACCACGT-3', ...

Embodiment 3

[0063] Example 3: PLDε-OE plants increase the absorption and utilization of N nutrients and seed yield

[0064] (1) Expression analysis of nitrogen transporter genes NRT1.1 and NRT2.1

[0065] In order to detect the absorption and utilization of N nutrients by PLDε, the expression levels of genes NTR1.1 and NTR2.1 related to N nutrient transport in plants were analyzed by Real-time. The data showed that: in severe nitrogen deficiency (0.3mM N) Under the same conditions, the expression levels of NTR1.1 in the leaves of OE24-2 and OE37-2 plants were significantly higher than those of the wild type, which were 4.5 and 1.5 times that of the corresponding WT plants, while those in the roots were 1.5 and 1.3 times, respectively. When plants were grown under the normal level of nitrogen supply (3mM N), the expression level of NTR1.1 in OE was 40 and 26 times that of wild type, and the expression level in roots was 0.7 and 1.9 times. Under nitrogen deficiency (0.3mM) conditions, the ex...

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Abstract

The invention belongs to the technical field of plant molecular breeding and biology, and discloses application of a PLD (phospholipase D) epsilon gene in increasing nitrogen nutrition effective utilization and seed yield of crops. The nucleotide sequence of the PLD epsilon gene is shown as SEQ ID NO:1, or has a similarity more than or equal to 60 percent as the nucleotide sequence shown as SEQ ID NO:1. The invention also discloses a method for increasing nitrogen nutrition effective utilization and seed yield of rape. Tests prove that growth and occurrence of lateral roots of rape can be promoted due to overexpression of AtPLD epsilon either in nitrogen starvation or nitrogen sufficiency condition, increase of rape leaf tissue cell growth and biological yield can be reinforced, absorption transportation and assimilation metabolism processes of rape plants can be promoted, which are particularly indicated in that the expression quantities of genes NTR1.1 and NTR2.1 related to overexpressed plant N transportation are remarkably higher than that of a wild type, and the nitrogen absorption rate is remarkably higher than that of the wild type.

Description

technical field [0001] The invention belongs to the fields of plant molecular breeding and biotechnology, and specifically relates to the application of PLDε gene in increasing the efficient use of crop nitrogen nutrition and seed yield. Background technique [0002] The metabolism of nitrogen nutrition is closely related to a variety of biochemical pathways, and it is also one of the essential elements for all living organisms. Plants absorb nitrates, nitrites and ammonium salts from the soil, transport and assimilate them for various metabolic pathways. Many studies have shown that plant root growth is closely related to the level of N nutrient supply. It was observed that when the concentration of exogenous nitrate was sufficient, the number of lateral roots of Arabidopsis thaliana did not change compared with that under normal conditions, but the length of lateral roots increased by 2 times compared with that under normal conditions, and this increase was attributed to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 鲁少平王学敏洪月云
Owner HUAZHONG AGRI UNIV
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