Application of maize Zm675 gene in plant quality improvement

A zm675 gene technology, applied in the field of biotechnology and genetic breeding, can solve the problems of reducing content, poor predictability, affecting the development of corn kernels, etc., and achieve the effect of increasing lysine and protein content and improving nutritional quality

Active Publication Date: 2019-01-11
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But there are still some defects and deficiencies in QPM corn: (1) Compared with common corn, the yield of QPM corn is low; low soil nitrogen, etc.) constraints
However, the characteristics of long breeding cycle, low efficiency and poor predictability have increased the difficulty of cultivating high-resistant QPM varieties.
(3) Since the o2 allele that provides QPM traits is recessive, when QPM corn is inoculated with normal endosperm corn pollen, the good genetic traits of QPM will be lost
However, the accumulation of excess free lysine will affect the development of corn kernels, and the instability of free lysine makes it easy to be damaged during processing (Wenefrida et al., 2013)
[0004] On the other hand, maize with higher lysine content can be obtained by inhibiting the expression of 19-kD or 22-kD α-gliadin and reducing the content of 19kD or 22kD gliadin in the grain (Segal et al.2003; Huang et al.2004; Huang et al.2005;) However, the grain phenotype of this corn is the same as the o2 mutant, producing floury endosperm, which limits its application in production (Wu and Messing, 2012)

Method used

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  • Application of maize Zm675 gene in plant quality improvement
  • Application of maize Zm675 gene in plant quality improvement
  • Application of maize Zm675 gene in plant quality improvement

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Cloning and sequence analysis of Zm675 gene

[0046] Total RNA was extracted from maize inbred line B73, and cDNA was synthesized by reverse transcription with Oligo dT as primer.

[0047] According to the maize genome sequence (maizeGDB: GRMZM2G051675), primers 675-F1 (SEQ ID NO.3): 5'-CACCAGAGGCGATGTCGTC-3' and 675-R1 (SEQ ID NO.4): 5'-TGGTGAGAGCATCTTCAGTC-3' were designed to cDNA as a template for PCR amplification. Reaction conditions: 95°C, 30sec; 58°C, 30sec, 72°C, 30sec, 30 cycles. The amplified product was ligated with the T vector pMD-19T, transformed into Escherichia coli DH5α, and the recombinant plasmid was obtained. Sequencing results show that the cloned Zm675 coding sequence is 669bp in length (as shown in SEQ ID NO.2), encoding 222 amino acid residues (as shown in SEQ ID NO.1), and the lysine content in the encoded protein is 18.56% (w / w).

Embodiment 2

[0048] Example 2 Construction of expression vector pSB130-675

[0049] Zm675 was excised from the above T vector with BamHI and HincII, the plant expression vector pSB130-F128 (ZL201310398102.9) was cut with BamHI and Sma I, and the Zm675 fragment was connected to the vector to construct a recombinant plasmid pSB130-675 ( figure 1 ). This plasmid includes two T-DNAs. T-DNA1 includes a Zm675 expression cassette driven by a millet seed-specific promoter F128 (CN101063139A); T-DNA2 includes an HPT (hygromycin phosphotransferase gene) expression cassette driven by a CaMV35S promoter. After the pSB130-675 vector was successfully constructed, it was transformed into Agrobacterium LBA4404 for the genetic transformation of maize. Agrobacterium transformation was carried out by conventional methods in the art.

Embodiment 3

[0050] The genetic transformation of the immature embryo of embodiment 3 maize

[0051] Preparation of Agrobacterium bacteria liquid: Agrobacterium LBA4404 (pSB130-675) was cultured on YEB solid medium (containing Sm 125 μg / mL, Kan 100 μg / mL) at 28°C for two days and collected the bacteria. Suspend the cells with infection medium to OD 600 After shaking the bacteria at 0.6-0.8, 28°C, 75rpm in the dark for 2-4 hours, dilute the shaken bacteria solution to OD with infection medium 600 Reserve for 0.3-0.4. Using maize inbred line 178 as the transformation recipient, the immature embryos (about 1.5-2.0 mm in size) 12 days after pollination of the 178 inbred line were stripped and infected with an Agrobacterium-free medium, soaked and washed twice. , Pour off the remaining medium, infect the immature embryos with the Agrobacterium solution prepared in advance for 15 minutes, then put the infected immature embryos on sterile filter paper and dry them, transfer them to the co-culti...

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Abstract

The invention relates to an application of a maize Zm675 gene in plant quality improvement. The invention discloses an application of a Zm675 gene, a Zm675 protein or an expression cassette or vectorcontaining the Zm675 gene in increase of lysine and/or protein content of plants. The Zm675 gene is cloned into maize, and the transgenic maize with the Zm675 gene is constructed. The content of lysine and protein in seeds of transgenic maize enhanced by Zm675 gene expression is significantly increased, and the number of protein bodies in endosperm cells is significantly increased. At the same time, the high protein and high lysine traits of transgenic maize transformed with Zm675 gene can be inherited stably. Zm675 gene has important application value for increasing the content of lysine andprotein in plant seeds and improving the nutritional quality of plants.

Description

technical field [0001] The invention relates to the fields of biotechnology and genetic breeding, in particular to the application of maize Zm675 gene in improving plant quality. Background technique [0002] Corn is an important food crop and feed crop, but the content of protein and some essential amino acids in corn kernels is low, which limits the nutritional value of corn to a certain extent. Lysine is the first limiting amino acid in corn grains, therefore, increasing the content of protein and essential amino acids in grains, especially lysine can improve the nutritional quality of corn. In 1964, the discovery of the opaque2 maize mutant opened a precedent for the improvement of maize nutritional quality. The O2 gene encodes a bZIP-like transcription factor that can regulate the synthesis of gliadin, and the mutation of the O2 gene leads to the lack of lysine in the maize endosperm. The proportion of gliadin in the endosperm decreased, and the proportion of lysine in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8251C12N15/8254
Inventor 赵倩李婉影于静娟朱登云
Owner CHINA AGRI UNIV
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