Gene for regulating and controlling corn drought stress resistance and application thereof
A corn and genetic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of difficult production and utilization
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Embodiment 1
[0046] Example 1 Overexpression of ZmPP2CA10 weakens drought tolerance in maize
[0047] The full-length CDS of ZmPP2CA10 gene primers were designed using the reference sequence of maize B73 as a template, and part of the vector sequence was added to both ends of the primers, and the fragment was cloned in vitro using the B73 leaf cDNA as a template, and connected to the overexpression vector by homologous recombination pZZ0153-3HA, and sequenced to verify that the correct clone was obtained. The overexpression vector plasmid was sent to China Seed Group Co., Ltd. (Wuhan) to obtain the transgenic plants of the gene. The applicant obtained two homozygous overexpression ZmPP2CA10 maize transgenic material lines 6 and 12 through herbicide screening.
[0048] Extract the RNA of transgenic material family 6 and family 12, and wild-type C01 material, perform reverse transcription, and detect whether the target gene is overexpressed at the transcriptional level by fluorescent quanti...
Embodiment 2
[0052] Example 2 Identification of ZmPP2CA10 Preference Alleles Associated with Drought Tolerance
[0053] In order to further confirm the details of the gene mutation of ZmPP2CA10, the applicant randomly selected 100 materials from different inbred lines of maize to resequence the ZmPP2CA10 gene. Using the genomic DNA of these 100 materials as a template, primers were designed and amplified based on the B73 reference genome sequence, and the ZmPP2CA10 promoter region and gene region (including exons and introns) were measured respectively.
[0054] Multiple sequence alignments were performed on the ZmPP2CA10 sequence of each material, and all the SNP / InDel sites and the survival rate data of the associated population (the data came from previous articles published by others) were used for candidate gene association analysis, and a site InDel- 338 were significantly correlated with drought ( figure 2 A).
[0055] There is an InDel-308 site near the InDel-338 site. Through l...
Embodiment 3
[0061] Example 3 Luciferase experiments verify that the ERSE element in the promoter region is necessary for the activation of ZmPP2CA10 by endoplasmic reticulum stress signals, but does not affect the activation of ZmPP2CA10 by the ABA pathway
[0062] Clone the 1000bp promoter fragment upstream of the ZmPP2CA10 gene ATG in the CIMBL55 material. The InDel-338 site of ZmPP2CA10 in this material lacks the ERSE element. This fragment is named A10pΔERSE. The nucleotide sequence of the missing part is shown in SEQ ID NO: 1. After mutation The nucleotide sequence of the promoter of ZmPP2CA10 is shown in SEQ ID NO:2. A point mutation was made to A10pΔERSE, and the InDel-338 site was mutated to contain an ERSE element, which was named A10pERSE. The two fragments were ligated by restriction enzymes and connected to the pGL-Basic-LUC vector to construct pGL-Basic-A10pΔERSE-LUC and pGL-Basic-A10pERSE-LUC. The two plasmids were transformed into maize protoplasts and cultured overnight, ...
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