Application of CDS (Coding Sequence) sequence of CBL9 (Calcineurin B-Like) gene of corn

Abstract

The invention relates to an application of a CDS (Coding Sequence) sequence of a CBL9 (Calcineurin B-Like) gene of corn. The invention provides the function of the CDS sequence of the CBL9 gene of corn for the first time. The CDS sequence of the CBL9 gene of corn is cloned to a plant expression vector and the vector is transferred to a plant body, so that the obtained transgenetic plant is saline-alkaline tolerant, osmotic stress resistant, high glucose resistant and ABA (Abscisic Acid) stress resistant. ZmCBL9 protein takes part in the salt tolerant and osmotic stress resistant process by regulating the downstream related gene and has important meaning for cultivating high yield transgenetic crops.

Description

technical field

[0001] The invention relates to the field of transgenic plants, in particular to the application of the CDS sequence of the corn CBL9 gene. Background technique

[0002] In nature, plants are different from animals, which cannot escape adversity but can only cope with it. Therefore, plants have evolved a set of fine-tuning mechanisms for sensing adversity stress, transmitting adversity signals, and responding at the molecular, cellular, and physiological levels. Many studies have shown that various stress environments will trigger Ca in plant cells 2+ change in concentration. Ca 2+ The change of concentration in time and space represents a special stress signal, called calcium signal; among them, calcium ion receptor plays a crucial role. To transduce signals and regulate the expression of downstream genes.

[0003] Ca in plants 2+ -Ca 2+ The sensor-actor-target gene is a key regulatory pathway. Many studies have shown that Ca 2+ Sensors can be divid...

Examples

Embodiment 1

[0031] Embodiment 1 Contains the construction of the expression vector of maize CBL9 gene CDS sequence

[0032]1. According to the sequence of the known target gene (GenBank: NM_001157847.1), design primers (forward and reverse primer sequences are shown in Seq ID No.3 and 4 respectively), amplify the ZmCBL9 gene CDS sequence from maize cDNA, and use Agarose gel kit to recover the target fragment.

[0033] 2. Ligate the recovered fragments with the pGEM-T vector, then transform DH5α competent cells, obtain positive monoclonals, send them for sequencing, and obtain monoclonals consistent with the original sequence after sequence comparison, shake the bacteria and extract the monoclonal plasmid , and named it pGEM-ZmCBL9.

[0034] 3. Digest pGEM-ZmCBL9 and p3301 plasmids with Nco I and BstE II, respectively, and connect them with T4 DNA ligase. Then the DH5α competent cells were transformed, and the positive monoclonal obtained was sent for sequencing. After the sequence align...

Embodiment 2

[0036] Example 2 Obtaining of Transgenic Plants Containing Maize CBL9 Gene CDS Sequence

[0037] 1. Transform the constructed overexpression vector p3301-ZmCBL9 into Agrobacterium GV3101.

[0038] 2. Transform wild-type Arabidopsis thaliana by floral dipping.

[0039] 3. Convert the T obtained after conversion 1 The generation seeds were vernalized for 3 days, and then directly sowed in nutrient soil for growth. After about two weeks of normal growth, spray T with 0.5‰ of PPT (phosphinothricin) 1 Transformed plants (ie transgenic plants with 35S::ZmCBL9 gene) were screened from Arabidopsis thaliana.

[0040] 4. Since the plant expression vector p3301 has the Basta gene of resistance to PPT (phosphinothricin), it can be transferred to the plant along with the target gene when transformed, so most of the untransformed plants die after spraying PPT (phosphinothricin), while Transformed plants can still grow normally. The transformed plants were harvested separately according ...

Embodiment 3

[0043] Example 3 PCR Identification of Transgenic Plants Containing Maize CBL9 Gene CDS Sequence

[0044] 1. Using the total DNA of the extracted positive plants as a template, use ZmCBL9 gene CDS sequence primers (Seq ID No.3-4) for PCR amplification. The positive plants can amplify a 642bp band, while the wild-type plants cannot amplify Add destination strip ( figure 2 ).

[0045] 2. Harvest each transgenic line T 2 After substituting the seeds, the seeds were disinfected with 0.5% NaClO. Then spot-planted in MS medium plates containing 0.5‰ of PPT (phosphinothricin) (about 50 seeds were needed for each transgenic line), and the wild type was used as a negative control.

[0046] 3. After 3 days of vernalization, place them in a light incubator at 22°C for growth, and observe the growth of the seedlings one week later. The wild type cannot survive in MS medium containing 0.5‰ PPT (phosphinothricin).

[0047] 4. T 2 Due to gene segregation and free combination in the ge...

technical field

[0001] The invention relates to the field of transgenic plants, in particular to the application of the CDS sequence of the corn CBL9 gene. Background technique

[0002] In nature, plants are different from animals, which cannot escape adversity but can only cope with it. Therefore, plants have evolved a set of fine-tuning mechanisms for sensing adversity stress, transmitting adversity signals, and responding at the molecular, cellular, and physiological levels. Many studies have shown that various stress environments will trigger Ca in plant cells 2+ change in concentration. Ca 2+ The change of concentration in time and space represents a special stress signal, called calcium signal; among them, calcium ion receptor plays a crucial role. To transduce signals and regulate the expression of downstream genes.

[0003] Ca in plants 2+ -Ca 2+ The sensor-actor-target gene is a key regulatory pathway. Many studies have shown that Ca 2+ Sensors can be divid...

Examples

Embodiment 1

[0031] Embodiment 1 Contains the construction of the expression vector of maize CBL9 gene CDS sequence

[0032]1. According to the sequence of the known target gene (GenBank: NM_001157847.1), design primers (forward and reverse primer sequences are shown in Seq ID No.3 and 4 respectively), amplify the ZmCBL9 gene CDS sequence from maize cDNA, and use Agarose gel kit to recover the target fragment.

[0033] 2. Ligate the recovered fragments with the pGEM-T vector, then transform DH5α competent cells, obtain positive monoclonals, send them for sequencing, and obtain monoclonals consistent with the original sequence after sequence comparison, shake the bacteria and extract the monoclonal plasmid , and named it pGEM-ZmCBL9.

[0034] 3. Digest pGEM-ZmCBL9 and p3301 plasmids with Nco I and BstE II, respectively, and connect them with T4 DNA ligase. Then the DH5α competent cells were transformed, and the positive monoclonal obtained was sent for sequencing. After the sequence align...

Embodiment 2

[0036] Example 2 Obtaining of Transgenic Plants Containing Maize CBL9 Gene CDS Sequence

[0037] 1. Transform the constructed overexpression vector p3301-ZmCBL9 into Agrobacterium GV3101.

[0038] 2. Transform wild-type Arabidopsis thaliana by floral dipping.

[0039] 3. Convert the T obtained after conversion 1 The generation seeds were vernalized for 3 days, and then directly sowed in nutrient soil for growth. After about two weeks of normal growth, spray T with 0.5‰ of PPT (phosphinothricin) 1 Transformed plants (ie transgenic plants with 35S::ZmCBL9 gene) were screened from Arabidopsis thaliana.

[0040] 4. Since the plant expression vector p3301 has the Basta gene of resistance to PPT (phosphinothricin), it can be transferred to the plant along with the target gene when transformed, so most of the untransformed plants die after spraying PPT (phosphinothricin), while Transformed plants can still grow normally. The transformed plants were harvested separately according ...

Embodiment 3

[0043] Example 3 PCR Identification of Transgenic Plants Containing Maize CBL9 Gene CDS Sequence

[0044] 1. Using the total DNA of the extracted positive plants as a template, use ZmCBL9 gene CDS sequence primers (Seq ID No.3-4) for PCR amplification. The positive plants can amplify a 642bp band, while the wild-type plants cannot amplify Add destination strip ( figure 2 ).

[0045] 2. Harvest each transgenic line T 2 After substituting the seeds, the seeds were disinfected with 0.5% NaClO. Then spot-planted in MS medium plates containing 0.5‰ of PPT (phosphinothricin) (about 50 seeds were needed for each transgenic line), and the wild type was used as a negative control.

[0046] 3. After 3 days of vernalization, place them in a light incubator at 22°C for growth, and observe the growth of the seedlings one week later. The wild type cannot survive in MS medium containing 0.5‰ PPT (phosphinothricin).

[0047] 4. T 2 Due to gene segregation and free combination in the ge...

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