Associated protein for regulating and controlling round-leaf characteristics of cucumbers as well as coding gene thereof and application thereof

A technology related to protein and round leaves, applied in the field of molecular genetics and breeding, can solve the problem of cucumber leaf shape change and other problems

Active Publication Date: 2018-06-12
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is evidence that PID controls the transport of polar auxin by regulating the subcellular localization of PIN protein. The abnormal expression of CsPID may change the steady-state level of auxin and cause serious defect of polar transport of auxin, which leads to the change of cucumber leaf shape.

Method used

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  • Associated protein for regulating and controlling round-leaf characteristics of cucumbers as well as coding gene thereof and application thereof
  • Associated protein for regulating and controlling round-leaf characteristics of cucumbers as well as coding gene thereof and application thereof
  • Associated protein for regulating and controlling round-leaf characteristics of cucumbers as well as coding gene thereof and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Phenotype and genetic analysis of embodiment 1 mutant

[0021] A cucumber round-leaf mutant rl was screened from the Changchun Mici mutant library induced by EMS (ethyl methane sulfonate). The mutant showed round leaves at the early stage of development, no calyx in both male and female flowers, sterile female flowers, asymmetric development of cotyledons (three cotyledons are the most common phenotype) and abnormal root development.

[0022] The mutant r1 was used as the male parent, and it was crossed with the wild-type Micila vinifera to obtain F1 plants of the first generation, and all F1 offspring had wild-type phenotypes. Since the homozygous r1 mutant plants are female sterile, wild-type plants (RLRL or RLr1) and mutant plants (rlr1) from the F2 generation of crosses between r1 mutants and WT wild-type were counted in 78 Only 24 plants in the F2 population of plants exhibited the round leaf phenotype, with a segregation ratio of 3:1 (χ 2 =0.19, P=0.66), indicat...

Embodiment 2

[0023] Embodiment 2 Location of CsPID gene

[0024]The round leaf mutant and wild-type leaf DNA of the F2 segregation population was extracted by conventional method-CTAB method. The DNA mixing pool was crushed to 150bp-250bp with an ultrasonic breaker, and the whole genome resequencing was performed on the small fragment DNA library constructed based on the Illumina HiSEQ 2500 sequencing platform. After removing low-quality reads through quality control, use BWA (version: 0.5.9rc1) and SAMtools software (version: 0.1.19) to compare the data with the cucumber genome sequence (“9930” reference genome), and divide the number of SNPs Calculate the allele index based on the sequencing depth of this site. The same SNP was screened out in the two mixed pools, and the corresponding allele index was subtracted to calculate the Δallele index. The same method was used to randomly perform high-throughput sequencing on a single wild-type Myacina changchun plant, and the SNP sites betwee...

Embodiment 3

[0025] Example 3 CsPID gene cloning and identification

[0026] The CsPID gene mutation is located at the second exon 1092bp of Csa1M537400, and the base G is mutated to A. In order to verify the sequencing results, the cDNA of the gene numbered Csa1M537400 in the Cucurbit Genomics Database was sequenced, and it was confirmed that the original guanine (G) of the cDNA in the coding region was mutated to adenine (A), resulting in the mutation of the encoded V (valine) to M (methionine), consistent with the sequencing results, confirmed that the mutation of this gene resulted in the production of a round-leaved calyx-less mutant, and the gene was named CsPID.

[0027] Obtaining the full-length cDNA of CsPID gene:

[0028] The total RNA of Cucumber radix and round leaves was extracted using TRIzol method, and DNA was digested at 25°C for 30 minutes, and 2 mg of total RNA was extracted as a template using the reverse transcription kit PrimeScript from TaKaRa Company TM RTreagent...

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Abstract

The invention discloses an associated protein for regulating and controlling round-leaf characteristics of cucumbers as well as a coding gene thereof and application thereof. An amino acid sequence ofthe protein is as shown in SEQ ID NO:2; protein kinase, which belongs to an AGC kinase family, of serine / threonine is coded by a PID gene; a coding gene of the protein is a base sequence as shown ina sequence table SEQ ID NO:1; guanine (G), at a site 1092bp, of the base sequence is substituted by adenine (A), Val (valine) at a site 346 of a coded amino acid residue sequence is mutated to be Met(methionine); the protein inhibits polar translocation of auxin, causes polar defect of auxin transport, has a blade edge smooth plant phenotype, has unapparent saw-tooth bugles, and has nearly roundleaves; a main root is not developed, and a main lateral root does not have obvious difference; male and female flowers are free of calyxes, and female flowers are not developed and the like. The geneand the protein can provide a basis for improving cucumber leaf type, increasing the yield, cultivating ideal plants and researching an auxin signal channel molecule regulation mechanism.

Description

technical field [0001] The invention relates to genetic engineering and cucumber proteins and genes, and belongs to the field of molecular genetic breeding, in particular to a protein related to the regulation of cucumber round leaf traits, its coding gene and its application. Background technique [0002] Cucumber (Cucumis sativus L.2n=2x=14) belongs to Cucurbitaceae Melon and is an annual vine herbaceous plant. Generally, the leaves are palmately 3-5 lobed, and the lobes are triangular and serrated. With the development of genomics, mutant material has become one of the direct and effective ways to study gene function. In the cucumber mutant library constructed by EMS, a stable hereditary single-gene controlled cucumber round-leaf mutant was found. The mutants exhibited smooth and nearly round leaf margins, no calyx, sterile female flowers, asymmetric development of cotyledon and abnormal root development. Leaf is the main organ of photosynthesis and respiration, and one...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54
CPCC12N9/12C12Y207/11
Inventor 娄群峰宋蒙飞程凤陈劲枫
Owner NANJING AGRICULTURAL UNIVERSITY
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