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Plant anther pollen development late-stage specific expression promoters and application thereof

A technology for pollen development and specificity, applied in the field of DNA application and isolated DNA, can solve the problems of long waiting time, high homology gene silencing, and plant growth and development effects.

Pending Publication Date: 2020-05-15
SHENZHEN INST OF MOLECULAR CROP DESIGN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some constitutive strong promoters are widely used in the field of agricultural biotechnology, such as the CaMV35S promoter of cauliflower mosaic virus and the Ubiquitin-1 promoter of maize. When it is expected to improve the quality of crops, the improvement effect is not obvious because the time (developmental stage-specific) or space (tissue-organ-specific) of the expression of the target gene is not well controlled, or because these constitutive promoters induce gene The expression level is too high to affect the growth and development of plants. These are the obstacles encountered when using constitutive strong promoters combined with functional genes to improve crop quality
[0004] In addition, when studying certain metabolic processes or regulatory pathways, it is often necessary to transform two or more genes on the same pathway into the same strain. If one of the genes is transformed to obtain a transgenic plant and then another gene is transformed, or After the two genes are transformed separately and then hybridized, it takes a long time to wait.
In order to improve efficiency and shorten the time for multiple gene transformation, it has recently been reported that new vectors can be used to transform multiple genes at the same time, but if the same promoter is used repeatedly in multigene transformation, it will also be difficult due to the high homology of the promoter sequence. Sex may lead to gene silencing

Method used

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  • Plant anther pollen development late-stage specific expression promoters and application thereof
  • Plant anther pollen development late-stage specific expression promoters and application thereof
  • Plant anther pollen development late-stage specific expression promoters and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Analysis of Tissue Expression Patterns of OsLPS5 and OsLPS6 Genes

[0049] The inventor obtained two highly expressed genes specifically in rice late pollen by RNA-Seq sequencing. The roots, stems, leaves, palea, palea, pistil, anthers (Stage 6-12) and endosperm (DAP 7 and DAP 25) of rice wild-type Nipponbare plants were taken respectively, RNA was extracted, reverse transcribed into cDNA as a template, Using rice ACTIN gene as an internal reference, primers for OsLSP5, OsLPS6 and ACTIN genes were designed respectively, and the expression of OsLSP5 and OsLSP6 genes in various tissues of rice was analyzed by Quantitative Real time-PCR (qRT-PCR).

[0050] Wherein, the qRT-PCR primers of OsLSP5 gene are: primer 1: 5'-TCGTTCTCGTCGTGATCCTCCTC-3' (SEQ ID NO: 5) and primer 2: 5'-GCAGATTCACTGCTGGACTCTTCC-3' (SEQ ID NO: 6); The qRT-PCR primers are: primer 3: 5'-GGCGGCATTGTTGGTCGTTGGCTCA-3' (SEQ ID NO: 7) and primer 4: 5'-TCTTGACAAGGAAGCGGACGGAGAA-3' (SEQ ID NO: 8); t...

Embodiment 2

[0056] Cloning of embodiment 2.BT1(TP)-ZM-AA1 fusion gene, pOsLSP5 promoter, pOsLSP6 promoter

[0057] Using the pZhen18 vector reported earlier as a template (Chang Zhenyi, Chen Zhufen, Wang Na, et al. (2016) Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility gene. Proceedings of the National Academy of Sciences of the United States of America 113,14145-14150), the primers required for cloning the BT1(TP)-ZM-AA1 fusion gene are:

[0058] Primer 7: 5'-CCGGGGATCC TCTAGA ATGGCGGCGACAATGGCAGTGACG-3' (SEQ ID NO: 11);

[0059] Primer 8: 5'-GGCCAGTGCC AAGCTT TCAAGGAAAAGACGTTATGCAGTG-3' (SEQ ID NO: 12);

[0060] The primers required for cloning the promoter pOsLSP5 are:

[0061] Primer 9: 5'-CCATGATTAC GAATTC GGCTGATAAATATAAGCATAAGCGAAAG-3' (SEQ ID NO: 13);

[0062] Primer 10: 5'-TCGCCGCCAT TCTAGA GGCCGATGTAGCTGTTGGTGCTCACCTA-3' (SEQ ID NO: 14);

[0063] The primers required for cloning the promoter pOsLSP6 ...

Embodiment 3

[0069] Example 3. Construction of expression vectors pSZYJY-14 and pSZYJY-15

[0070] Such as figure 2 As shown, the BT1(TP)-ZM-AA1 fusion gene fragment recovered by PCR glue in Example 2 was connected under the effect of homologous recombinase In-fusion into the vector pCAMBIA1300 double-digested with XbaI and HindIII, and picked The colonies were detected by PCR, and the colonies with positive PCR results were selected for sequencing and sequencing to verify that they were correct, and the corresponding positive clone plasmid was extracted and named pSZYJY-13.

[0071]Then, the pOsLSP5 or pOsLSP6 promoter sequence recovered from the glue in Example 2 was connected under the action of homologous recombinase In-fusion into the vector pSZYJY-13 cut with EcoRI and XbaI again, and colonies were picked for PCR detection , select the colonies with positive PCR results for sequencing, and after the sequencing verification is correct, extract the corresponding positive clone plasmi...

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Abstract

The invention discloses plant anther pollen development late-stage specific expression promoters pOsLSP and application thereof, belongs to the technical field of plant biology, and particularly relates to application of rice anther pollen development late-stage specific expression promoters in regulation of male sterility of rice. Nucleotide sequences of pOsLSP5 and pOsLSP6 promoters of rice Nipponbare are cloned and embedded with an amyloplast transport signal peptide gene BT1 (TP) and an alpha-amylase gene ZM-AA1 respectively to construct a recombinant expression vector and perform genetictransformation. Under the drive of the promoters pOsLSP5 and the guide of the amyloplast transport signal peptide, the alpha-amylase gene can be expressed in late-stage pollen and hydrolyze starch tocause transgenic pollen sterility, and the promoters pOsLSP have high accuracy, can be applied to maintenance of homozygous recessive state of male sterile plants and mass expanding propagation of sterile lines, omit an artificial castration step in a hybrid seed production process, and have broad application prospects in crop planting resource improvement, genetic breeding and other aspects.

Description

technical field [0001] The present invention belongs to the field of plant biotechnology, in particular, the present invention relates to isolated DNA, which can direct the specific transcription and / or expression of nucleic acid operably linked downstream of it in plant anther pollen. In addition, the present invention also relates to expression cassettes and plants containing the DNA, and to applications of the DNA. Background technique [0002] Plant gene regulation is mainly carried out at the transcriptional level, which is coordinated by a variety of cis-acting elements and trans-acting factors. The promoter is an important cis-acting element. It is a DNA sequence located in the upstream region of the 5' end of the structural gene to regulate gene transcription. It can activate RNA polymerase to accurately combine with the template DNA to ensure accurate and efficient transcription. play a key role in transcriptional regulation. According to the different characteris...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/02A01H4/00A01H6/46
CPCC07K14/415C12N15/8231C12N15/8245C12N15/8289A01H4/00
Inventor 王梦龙唐晓艳
Owner SHENZHEN INST OF MOLECULAR CROP DESIGN
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