Transgenic element and application thereof, method for differentiating male sterility line and fertile maintainer line, and expanding propagation method of male sterile line of maize

A male sterility gene and genetically modified technology, applied in the agricultural field, can solve the problems of unfavorable mechanized differentiation, difference in grain shape and size, and decreased accuracy of differentiation, and achieve good application value, increase purity, and reduce volume.

Inactive Publication Date: 2015-05-13
ZHEJIANG XINAN CHEM INDAL GROUP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the difference of corn sowing season and growth environment, there will also be differences in the shape and size of the kernels, which is not conducive to the mechanized distinction of large quantities, and the accuracy of the distinction will also decrease accordingly
Therefore, there are certain limitations in the production and application of using this trait as a grain marker

Method used

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  • Transgenic element and application thereof, method for differentiating male sterility line and fertile maintainer line, and expanding propagation method of male sterile line of maize
  • Transgenic element and application thereof, method for differentiating male sterility line and fertile maintainer line, and expanding propagation method of male sterile line of maize
  • Transgenic element and application thereof, method for differentiating male sterility line and fertile maintainer line, and expanding propagation method of male sterile line of maize

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1 Amplification of Ms8 gene promoter sequence

[0085] The present invention takes the maize male sterile mutant Ms8 as an example to describe the embodiment in detail. By analyzing the genome sequence of Ms8, it was found that the promoter region was located 906bp upstream of the transcription start site. Taking the genome sequence of the corn inbred line B73 as a reference, the upstream and downstream primers for amplifying the promoter sequence were designed. The primer sequence Ms8-Pro-R1:5'-ggatccGCAGGCTATGCAGCTTCTCT-3' (as shown in SEQ ID NO: 7), and a Pst I restriction site was added to the 5' end of the upstream amplification primer, and the downstream amplification primer A BamH Ⅰ restriction site was added to the 5', and the length of the amplified fragment was 2343bp (Sequence ID No:2)( Figure 4 ). The PCR reaction system is (20 μl): 1 μl of B73 genomic DNA, 10.5 μl of primer Ms8-Pro-F, 0.5 μl of primer Ms8-Pro-R1, 0.5 μl of dNTP, 2 μl of 10×PC...

Embodiment 2

[0086] Example 2 Amplification of Ms8 Gene Coding Sequence

[0087] In the maizegdb database (www.maizegdb.org), the coding sequence of the Ms8 gene was found to be 1239bp (Sequence ID No: 1). Taking the sequence of the maize inbred line B73 as a reference, primers were designed to amplify the coding sequence of the Ms8 gene ( Figure 5 ), the upstream primer sequence Ms8-CDS-F1: 5'-ggatccATGCTCCAGCTGCTGCGC-3' (as shown in SEQ ID NO: 8), the downstream primer sequence Ms8-CDS-R1: 5'-gagctcTCATGTGGCGGCGTTCCA-3' (as shown in SEQ ID NO : 9), and a BamH I restriction site was added to the 5' end of the upstream primer, and a Sst I restriction site was added to the 5' end of the downstream primer. The PCR reaction system is (20 μl): 1 μl of B73 cDNA, 0.5 μl of primer Ms8-CDS-F1, 0.5 μl of primer Ms8-CDS-R1, 0.5 μl of dNTP, 2 μl of 10×PCR Buffer, 0.2 μl of Taq enzyme, ddH 2 O 15.3 μl. PCR reaction conditions: 94°C for 5min, 94°C for 35S, 58°C for 40S, 72°C for 1.5min, 35 cycles, 7...

Embodiment 3

[0088] Embodiment 3 Amplification of Bt2 gene promoter sequence

[0089] The expression of the Bt2 interference fragment in the present invention uses the promoter of the gene itself, and the expression of the Bt2 gene is mainly concentrated in the endosperm of corn seeds, so the mRNA that utilizes the promoter to initiate transcription mainly exists in the seed endosperm. The genome sequence analysis of the Bt2 gene found that the promoter region was located 1271bp upstream of the transcription initiation site. Using the genome sequence of the maize inbred line B73 as a reference, primers were designed to amplify the promoter region of the Bt2 gene. The upstream primer sequence Bt2-Pro -F1: 5'-ctgcagTTTTCTCTCTCCCGCATGTT-3' (as shown in SEQ ID NO: 10), the downstream primer sequence Bt2-Pro-R1: 5'-tctagaATGGGGAATTCGAGGAAAGT-3' (as shown in SEQ ID NO: 11), and in A Pst Ⅰ ​​restriction site was added to the 5′ end of the upstream primer, and an Xba Ⅰ restriction site was added t...

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Abstract

The invention relates to the technical field of agriculture and in particular relates to a transgenic element and an application thereof, a method for differentiating a male sterility line and a fertile maintainer line, and an expanding propagation method of the male sterile line of maize. A functional nucleotide sequence controlling the male fertility of the maize and a nucleotide sequence controlling starch synthesis are transferred into the maize together, and then backcross transformation is performed to obtain a hybrid maintainer line. As the nucleotide sequence controlling starch synthesis in the maintainer line inhibits the starch synthesis and increase the sugar content to lead to kernel shrinking, the sterility line and the maintainer line can be doubly differentiated from quality and appearance, and the accuracy is improved. The male sterility line and the fertile maintainer line of the hybrid female parent of the maize can be differentiated accurately and efficiently by use of the method, and then the male sterility line of the hybrid female parent can be applied to expanding propagation and applied to the production of hybrid seeds. Meanwhile, the difference in quality and appearance is more prone to mechanical identification, and therefore, the working efficiency can be greatly improved.

Description

technical field [0001] The invention relates to the field of agricultural technology, in particular to a transgenic element and its application, a method for distinguishing a female male sterile line and a maintainer line of a corn hybrid, and a method for multiplying a female male sterile line of a corn hybrid. Background technique [0002] Corn is the food crop with the highest total output in the world. my country is the second largest corn producer after the United States. In 2012, my country's corn production surpassed rice and became my country's largest food crop. Corn is also an important ration, feed grain, industrial raw material and energy raw material, and plays a pivotal role in national production. [0003] Maize is mainly planted with hybrids, which inherit the dominant and beneficial genes of their parents, and their environmental adaptability such as disease resistance, insect resistance, and drought resistance has increased significantly, and their yield an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/29C12N15/113A01H1/02A01H5/00
Inventor 何军光吕洪坤金少军赵传慧楼亿圆张晓丽吴涛
Owner ZHEJIANG XINAN CHEM INDAL GROUP
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