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Construction method of high-lysine protein gene SB401 expression vector and application thereof

A high-lysine protein and high-lysine technology, which is applied in the field of construction of high-lysine protein gene Sb401 expression vector, can solve the problems of inactivity of inserted gene, low gene expression level and low conversion rate, etc. The effect of less gene silencing, short transformation period and low copy number

Inactive Publication Date: 2011-05-25
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the different promoters used in plant expression vectors, or the different methods of genetic transformation, resulting in low levels of gene expression, the nature of recombinant DNA molecules composed of foreign genes and vectors, and the host cells are also different. The specific steps for introducing recombinants into host cells method has many advantages, but also has some disadvantages
For example, the randomness of gene gun bombardment leads to low transformation rate; gene gun insertion is often multi-copy clustered integration into the recipient genome, and rearrangement may occur in multiple ways; homologous sequences can be DNA-DNA, DNA- RNA, RNA-RNA interaction, leading to transcriptional or post-transcriptional gene silencing; the bombardment process may also cause breakage of foreign genes, and make the inserted gene an inactive fragment; non-transformants or chimeras appear The possibility of higher and the cost of gene gun transformation is higher, etc.

Method used

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  • Construction method of high-lysine protein gene SB401 expression vector and application thereof
  • Construction method of high-lysine protein gene SB401 expression vector and application thereof
  • Construction method of high-lysine protein gene SB401 expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Hipramine protein gene SB401 expression carrier construction method, the specific steps are as follows:

[0029] 1. Select fresh potato pollen materials 0.1g, and use the Plant RNA MIDI KIT kit (purchased from Shanghai Biological Engineering Co., Ltd.) to extract potato high lyricine gene SB401 total RNA

[0030] 2. Reverse transcription

[0031] The first chain CDNA synthesis uses Promega's M-MLV reversal transcription enzyme mixing the following reagents in the 0.5ml centrifugal pipe:

[0032] 5 μg RNA sample

[0033] 2ML Oligo (DT) 15 (250ng / ml)

[0034] H 2 O

[0035] 28ml Total

[0036] After the sample is mixed, brief centrifugation, 65 ° C degeneration 5min, quickly place it on the ice for 10 minutes, after a short centrifugal, then add the following reagents to the tube:

[0037] 8ml 5 × First-Strand Buffer

[0038] 2ml dntp (10mm)

[0039] 1ml RNA enzyme inhibitor

[0040] 1ml reverse transcription enzyme (200units / ml)

[0041] 12ml topal

[0042]Gently m...

Embodiment 2

[0084] Example Two corn Agriculture transformation

[0085] This experiment adopts a root tumor Agrobus media to reorganize the hypertropin gene SB401 fragment CUB-SB401 to include infection, recovery, selection, regeneration, and seedlings after the infection, recovery, selection, regeneration, and seedlings.See Figure 4 To.The specific steps are as follows:

[0086] 1. Agrobus infection young embryos Put the newly peeled young embryo in a centrifugal tube containing D-INF (2ml), about 20 to 100 young embryos per tube, soak it with such a medium for more than 1h, and then filter out the out of the filtering out.D-INF, add 1 to 1.5ml of Agentobacteria liquid with a specific concentration (OD600 = 0.3 ~ 0.4), gently upside down the corruption 20 times, and then placed in the dark box for 5 minutes to ensure that all the young embryos are soaked in Agrobus liquidIn the whole process, avoid vortex oscillations. After five minutes, the young embryo and bacterial fluid are poured into ...

Embodiment 3

[0093] Example three rotor corn plants PCR detection

[0094] When the transplanting plant that is transplanted in the Example II is 7 to 8 leaves, the blades are extracted to extract DNA, and PCR technology is used to detect whether there is an exogenous gene; the plant DNA extraction method is performed by the CTAB proposed by Saghai-Maroof;PCR technology detects external genes; Figure 5 It shows the PCR detection results of the genetic SB401 of the T0 generation transformation.To SB401-8.The PCR detection primers used are (such as SEQ ID No.5, SEQ ID No.6):):

[0095] F2- SB401 : 5'-cacgcagttgcaacggaacg-3 '

[0096] R2- SB401 5'-ionaaCactCTTTTCTCCCAT-3 '

[0097] Destination fragment size: 560 BP, annealing temperature of 58 ° C, 35 cycles.

[0098]The extraction steps of the corn plant DNA are as follows:

[0099] 1. Take about 1cm 2 The left and right fresh young and tender leaves are placed in the 1.5ml EPPENDORF tube, and then the leaves with leaves are inserted in the ice ...

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Abstract

The invention relates to construction of a high-lysine protein gene SB401 expression vector and application thereof and aims to solve the technical problems of efficient introduction of an exogenous high-lysine protein gene into a plant and efficient expression of the protein gene. The method comprises the following steps of: extracting ribonucleic acid (RNA) of a potato, obtaining full-length complementary deoxyribonucleic acid (CDNA) of the potato through reverse transcription, obtaining a sb401 gene by a polymerase chain reaction (PCR) method, recombining the gene and cloning the recombined gene into a plant dual-element expression vector; and introducing the gene into a corn gene group by an agrobacterium-mediated method, obtaining an expression transgenosis positive seedling and performing efficient expression so as to increase the lysine content of corn and further enhance the quality of corn. The method has unique advantages of low cost, small copy number, high repeatability, a small number of gene silencing phenomena, short transformation period, capability of transforming large fragments and the like.

Description

Technical field [0001] The invention involves the field of gene biological engineering, which specifically involves the construction method and application of a hyperinoproteine gene SB401 expression carrier. Background technique [0002] Corn is an important food crop in my country.With the deepening of corn output and resistance, people are increasingly increasing the quality of corn rice.The content of protein and amino acids is an important indicator of quality.Increasing the content of protein and lysine in corn will increase the biological value of corn, and promote the digestion and use of human and animals of their protein.Corn is an important food crop in my country.With the deepening of corn output and resistance, people are increasingly increasing the quality of corn rice.The content of protein and amino acids is an important indicator of quality.Increasing the content of protein and lysine in corn will increase the biological value of corn, and promote the digestion a...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29
Inventor 铁双贵岳润清朱卫红齐建双王延召孙静卢彩霞柏松
Owner HENAN ACAD OF AGRI SCI
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