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Miniaturization cucumber plant associated protein and coding gene thereof and application thereof

A technology related to proteins and plants, applied in the field of molecular genetics and breeding, can solve problems such as lack of materials, obstacles to the rapid development of cucumber functional genomics and genetic research, and narrow genetic basis

Inactive Publication Date: 2017-10-17
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the geographic distribution of cucumber is very wide, its genetic base is narrow, resulting in a lack of available materials for various phenotypic gene studies, which hinders the rapid development of cucumber functional genomics and genetic research.

Method used

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  • Miniaturization cucumber plant associated protein and coding gene thereof and application thereof
  • Miniaturization cucumber plant associated protein and coding gene thereof and application thereof
  • Miniaturization cucumber plant associated protein and coding gene thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Phenotype and genetic analysis of embodiment 1 mutant

[0024] A cucumber plant miniaturized mutant MP1 was screened from the EMS (ethyl methylsulfonate) mutant library of Mici changchun. Compared with the wild type, the mutant has significantly reduced plant height, weakened growth vigor, reduced fertility, smaller and darker green leaves, no side branches, shorter tendrils, shorter fruit, and smaller rounded seeds ( figure 1 , 2 , 3, 4).

[0025] The mutant MP1 was used as the male parent and crossed with the wild-type Micila changchun to obtain F1 plants of the first generation. Compared with the wild-type, the plant height of the F1 plants became shorter, and the fruit length became shorter, showing an intermediate type. In the F2 obtained by self-pollination of the F1 generation, the segregation ratio of normal plants, intermediate types and mutants is 113:255:117≈1:2:1, and the normal plants are incompletely dominant mutations to the mutants, indicating that the...

Embodiment 2

[0026] The location of embodiment 2MP1 gene

[0027] The mutant and wild-type individuals in the obtained F2 population were used for mixed pool sequencing. The mutant DNA pool was randomly interrupted by ultrasonic method, and the library was constructed according to the method suggested by the Illumina Paired-End DNA Sample Prep kit. Then select fragments with a length between 200-300bp, and use the Hiseq 2000 platform (PE101) for resequencing. After the quality control of the raw sequencing data, low-quality data and joint contamination data were removed, the data was compared to the cucumber reference genome 9930 through SOAP2 software, and the reads aligned to the unique position were obtained. Then, using these data, SOAP SNP software was used to find the single nucleotide polymorphism (SNP) between the mutant and the 9930 reference genome, and a map of the mutant pool was made on the chromosome according to the SNP index. The same method was used to sequence and analyz...

Embodiment 3

[0028] Embodiment 3MP1 gene cloning and identification

[0029]The MP1 gene mutation is located 1058 bp away from the start codon, and the base C is mutated to T. In order to verify the sequencing results, the cDNA of the gene numbered Csa7M435510.1 in the Cucurbit Genomics Database was sequenced, and it was confirmed that the cytosine (C-1058) of the cDNA in the coding region was mutated to thymine (T), resulting in the encoded serine (Ser-353 ) was mutated to phenylalanine (Phe), which was consistent with the sequencing results. It was confirmed that the mutation of this gene led to miniaturization of plants, and it was named gene MP1.

[0030] Obtaining the full-length cDNA of MP1 gene:

[0031] Total RNA was extracted from the leaves of Cucumber Miciens and MP1 using the TRIzol method, and the DNA was digested at 25°C for 30 minutes, and 2 mg of the total RNA was extracted as a template using the reverse transcription kit PrimeScript from TaKaRa Company TM RTreagent Kit...

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Abstract

The invention discloses a miniaturization cucumber plant associated protein and a coding gene thereof and an application thereof. An amino acid sequence of the protein is shown as a sequence table 2, which belongs to AAA ATP enzyme family, and is related with microtubule associated protein; the coding gene of the protein has a base sequence shown as a sequence table 1, if the mutation is generated on the base sequence, such as cytosine (C) with 1058 bp bit is substituted by thymidine (T), serine (Ser) with 353 bit for coding the protein is changed to phenylalanine (Phe), the protein inhibits the normal growth of the microtubule cell, the microtubule cytoskeleton is lost, the arrangement is abnormal, concretely, the plant phenotype is plant miniaturization, the height of the mutant plant is about 20% of the height of a normal wild type plant, the fruit length is about 10% of the length of normal wild type fruit, and the leaves and the seeds are obviously shortened. The application of the protein and the coding gene can create condition for cucumber strain-type improvement and cross breeding or exploitation of transgene plants.

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 miniaturized cucumber plant-related protein and its coding gene and application. Background technique [0002] Cucumber (Cucumis sativus L.2n=2x=14) belongs to Cucurbitaceae Melon, an annual vine herb, and is one of the top ten vegetable crops in the world. Although the geographic distribution of cucumber is very wide, its genetic basis is narrow, resulting in the lack of available materials for various phenotypic gene studies, which hinders the rapid development of cucumber functional genomics and genetic research. In the cucumber mutant library constructed by EMS, a stably inherited single gene controlling cucumber plant miniaturization mutant was found. The mutant can be used to breed excellent new varieties of cucumber, and is also an ideal material for studying the genetics and evolution of cucum...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55A01H5/00
CPCC12N9/14C12N15/8261
Inventor 娄群峰付文苑陈劲枫
Owner NANJING AGRICULTURAL UNIVERSITY
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