CN111518803A - An RNAi fragment and its application for regulating lignin synthesis - Google Patents

Abstract

The invention provides an RNAi fragment and its application for regulating lignin synthesis, belonging to the technical field of plant genetic engineering. The improved DNA sequence shown in SEQ ID NO. , whose sequence is shown in SEQ ID NO.2. The RNAi fragment was applied to transgenic plants, and it was proved that the RNAi fragment could regulate the synthesis of lignin. By studying the CAD gene sequence of Eucalyptus urophylla, the present invention helps to promote the genetic engineering breeding of Eucalyptus urophylla transgenic directional regulation of lignin synthesis.

Description

An RNAi fragment and its application for regulating lignin synthesis

technical field

The invention belongs to the technical field of plant genetic engineering, in particular to an RNAi fragment and its application for regulating lignin synthesis.

Background technique

Guangxi's eucalyptus research is in a leading position in my country. It has been introduced in the early 1970s. After years of introduction experiments and hybrid breeding, many excellent tree species, excellent provenances and excellent families have been obtained. Eucalyptus urophylla clone GLU4 is an excellent eucalyptus species widely planted in Guangxi. Because of its cellulose content, cellulose length and other pulping indicators, it is significantly better than other eucalyptus species, and it has become the preferred tree species for pulp wood afforestation. Starting from the production demand of pulp wood, it is hoped that the lignin content in wood is lower, so the directional regulation of lignin synthesis has become the main research strategy for improving the quality of pulp wood. But so far, there is no report of CAD gene sequence in Eucalyptus urophylla, and genetic engineering regulation using the gene sequence has not been carried out.

SUMMARY OF THE INVENTION

In order to solve the above technical problems, the present invention provides an RNAi fragment and its constructed plant expression vector, host cell, etc. in the application of plant lignin monomers for regulating lignin synthesis, so as to effectively regulate lignin synthesis in a targeted manner, It plays an important role in genetic engineering research on the regulation of lignin synthesis.

To achieve the above object, the present invention provides the following technical solutions:

An RNAi fragment is composed of an improved DNA sequence derived from the CAD gene of Eucalyptus urophylla in a "tail-to-tail" manner; the improved DNA sequence has the DNA sequence shown in SEQ ID NO.1.

SEQ ID NO. 1tggttgagtgccgcagaagctgtcgcccttgcaattccgaccag

Further, the DNA sequence of the RNAi fragment is shown in SEQ ID NO.2.

SEQ ID NO.2

tggttgagtgccgcagaagctgtcgcccttgcaattccgaccagctggtcggaattgcaagggcgacagcttctgcggcactcaacca

The present invention provides a recombinant plant expression vector, comprising the RNAi fragment.

Further, the recombinant plant expression vector is a recombinant vector pCAMBIA3301-S2 constructed on the basis of pCAMBIA3301.

The present invention provides a host cell comprising the RNAi fragment or the recombinant plant expression vector.

Further, the host cells are Escherichia coli, Agrobacterium or plant cells. Such as Escherichia coli JM109 strain, Agrobacterium LBA4404 strain.

The present invention provides an application of RNAi fragment derived from Eucalyptus urophylla CAD gene sequence, which is to apply the RNAi fragment, the recombinant plant expression vector, or the host cell to reducing the synthesis of plant lignin.

Further, the RNAi fragment or the recombinant plant expression vector is transformed into a plant to directionally reduce the lignin synthesis of the plant.

Further, the host cell is used to infect a plant. The effect of reducing the lignin content of plants.

The present invention has the following beneficial effects:

1. The present invention abandons the idea of using full-length genes in traditional research strategies, and carries out bioinformatics analysis of the Eucalyptus urophylla CAD gene sequence, according to the prediction of the structural domain of the encoded protein and the analysis of the importance of the structural domain to the enzymatic activity. , and selected one of the shorter DNA sequences, which encodes a protein containing a Zn-binding site that is very important for CAD enzymatic activity. At the same time, according to the results of homology comparison and analysis of terrestrial plant CAD sequences, the bases were improved to obtain the improved DNA sequence: SEQ ID NO. 1tggttgagtgccgcagaagctgtcgcccttgcaattccgaccag.

2. The improved DNA sequence obtained by the present invention is used to construct RNAi fragments, which can efficiently identify the target fragment of the Zn binding structure site in the CAD gene, and induce RNA interference, inhibit the expression of the CAD gene, and reduce the activity of the CAD enzyme in plants.

3. The present invention only uses shorter DNA sequences "tail-to-tail" to form RNAi fragments in application, and commercializes target RNAi fragments by means of sequence synthesis, avoiding the construction of restriction endonucleases in traditional research strategies The process greatly simplifies the experimental steps and shortens the experimental period.

4. The present invention detects the lignin content of the RNAi fragment-transformed tobacco plants, and the results show that the lignin content in the transgenic tobacco plants is 11.44% lower than that of wild-type tobacco, which verifies the regulatory effect of the RNAi fragment on lignin synthesis and proves that RNAi fragments have the effect of inhibiting lignin synthesis, which can reduce the lignin content of plants.

Description of drawings

Fig. 1 is a photograph of the stem section of the wild-type plant in the embodiment of the present invention.

Figure 2 is a photograph of a stem section of a transgenic plant in the example of the present invention.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The % in the following examples, unless otherwise specified, are all mass percentages. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

Example 1. Improved DNA sequence

Blastp, ProtParam, psipred, SWISS-MODEL bioinformatics software was used to analyze the CAD gene sequence of Eucalyptus urophylla, and a candidate DNA sequence encoding protein sequence rich in Zn-binding structural sites was obtained by screening (SEQID NO.3: tggttgggtgccgcagaagctgtggcccttgcaattcggaccag) , based on this candidate sequence, use online tools such as Blastn to match the candidate sequence with the terrestrial plant CAD gene sequence, and improve the candidate sequence according to the results of sequence homology and consistency analysis, and obtain a 44bp sequence S-2 (SEQ ID NO. 1), the sequence information is: 5'-tggttgagtgccgcagaagctgtcgcccttgcaattccgaccag-3'.

Example 2. Construction of RNAi fragments

According to the sequence S-2 of Example 1, the inverted repeat sequence S2 (SEQ ID NO.2) is designed in a "tail-to-tail" manner, and the sequence information is:

5'–tggttgagtgccgcagaagctgtcgcccttgcaattccgaccagctggtcggaattgcaagggcgacagcttctgcggcactcaacca-3'.

Example 3. Construction of RNAi expression vector

After adding XhoI restriction sites at both ends of the sequence S2 described in Example 2, the sequences were synthesized by a bio company to obtain the cloning vector pUC57-S2 carrying the target fragment.

Take the overnight culture of Escherichia coli carrying the pUC57-S2 plasmid, and use a spin-column-type ordinary plasmid mini-extraction kit (Tiangen) to complete the plasmid extraction. The plasmid was digested with XhoI enzyme, and the digested product was detected by agarose gel electrophoresis, and the target sequence S2 band of about 100 bp was cut out and recovered with Universal DNA purification and recovery kit (Tiangen). At the same time, the plasmid pCAMBIA3301 was digested with XhoI enzyme to obtain the vector backbone with the bar gene sequence removed. The vector backbone was connected to the target sequence S1, and the ligation product was transformed into DH5a. The sequencing primer 3301-F (5'-CCCTTATCTGGGAACTACTCAC-3') on the vector was used. /3301-R (5'-CGCTGAAATCACCAGTCTCTC-3') PCR identification of transformants, construction of the correct vector can be amplified to obtain a specific band of about 200bp, the correct length of the cloned and extracted plasmid is sequenced, the sequencing results are consistent with the target Sequence alignment, the sequence is consistent to construct a successful RNAi vector, named pCAMBIA3301-S2.

Embodiment 4. Utilize RNAi fragment to regulate and control tobacco lignin synthesis

Using conventional methods, the recombinant vector pCAMBIA3301-S2 in Example 3 was introduced into Agrobacterium LBA4404, and positive strains were obtained through antibiotic plate screening, PCR identification and sequencing identification. According to the conventional method, Agrobacterium liquid infects tobacco leaves, and after co-cultivation, differentiation, seedling strengthening, rooting, transplanting and other stages of culture, transgenic tobacco seedlings are obtained.

Eight weeks after transplanting, the leaves of the seedlings were collected to extract total DNA, and the seedlings were identified by PCR with specific primers 3301-F/3301-R, and plants with a band of about 200bp could be successfully amplified, and the PCR products were further sequenced. The results were compared with the target sequence, and the plants with the same sequence were identified as positive transgenic plants.

The growth of the transgenic plants is not significantly different from that of the wild type. The leaves of the plants are unfolded, the leaves are green and the stems are straight. Samples were collected at 8 months of age for phenotypic testing, including the following:

(1) Detection of target gene expression level

Using tobacco actin gene (EU938079) as the internal reference gene and tobacco CAD gene (X62343.1) as the target gene, fluorescent quantitative detection primers were designed to detect the influence of RNAi fragments on the expression level of the target gene. The primer sequences are as follows:

actin-F CTGGAATCCATGAGACTACTTACAA;

actin-R AACCGCCACTGAGCACAATA;

CAD-F: GTATGGCACCAGAACAAGCAG;

CAD-R: CCAATGCCTCTTGTCTCTTCTTAT.

The leaf tissue was collected from the 5th section from the shoot tip of the transgenic tobacco plants, and wild-type tobacco was used as a control. RNA prep Pure polysaccharide and polyphenol plant total RNA extraction kit was used for RNA extraction, and then RNA LA PCR reverse transcription reagent was used. Cassette to synthesize cDNA. Gene expression levels were detected by fluorescence quantitative PCR using ABI SybrGreen PCR Master Mix (2X) kit. The detected tobacco CAD gene expression levels are shown in Table 1.

Table 1: Analysis of CAD Gene Expression Levels in Transgenic Tobacco

Plant number Relative expression levels of tobacco CAD genes Wild type 1.0131±0.0125 transgenic plants 0.1270±0.0267

As can be seen from Table 1, when the transgenic plants were tested, the expression level of tobacco CAD gene was detected to be reduced to 12.71% of the wild type, indicating that the transferred RNAi fragment significantly inhibited the expression of tobacco CAD gene as expected.

(2) Detection of lignin content in stems

The control and transgenic tobacco plants were selected respectively, the top of the stem was selected from the 3rd to the 10th node, the leaves were removed, the stem was cut into small sections, placed in an oven at 60°C for 24 hours, and crushed to 150 mesh. Sieve for acid washing lignin content detection. The content of acid washing lignin was determined by the method of GB/T 20805-2006. The test results are shown in Table 2.

Table 2: Lignin Content in Transgenic Tobacco

Plant number Lignin content (%) Wild type 13.37±0.04 transgenic plants 11.84±0.05

It can be seen from the test results in Table 2 that the lignin in the transgenic plants is reduced by 11.44%.

(3) Changes in stem diameter and xylem thickness of transformed plants

The 9-month-old tobacco stems were taken from the top of the 6th node, and cross-sectioned according to conventional methods to make paraffin sections, which were stained with safranin-fast green method, and the stem diameter and xylem thickness were measured according to the sections. The wild-type plant section is shown in FIG. 1 , and the transgenic plant section is shown in FIG. 2 . The specific data are shown in Table 3.

Table 3 Changes in stem diameter and xylem thickness of transformed plants

According to Figure 1, Figure 2, and the measurement results in Table 3, the measurement results of stem diameter between the transgenic line and the wild type showed no significant difference, but the xylem thickness of the transgenic plant was reduced by 7.61% compared with the wild type. The regulation effect of the RNAi fragment of the present invention on lignin synthesis is described.

Embodiment 5, the recombinant vector is transformed into plant regulation tobacco lignin synthesis

After the plasmid was extracted from the pCAMBIA3301-S2 recombinant vector in Example 3, it was mixed with gold powder according to conventional methods, and the tobacco callus was bombarded with a gene gun. According to the conventional method, the transgenic tobacco seedlings are obtained after the stages of hypertonic culture, recovery culture, differentiation, seedling strengthening, rooting, and transplanting.

Eight weeks after transplanting, the leaves of the seedlings were collected to extract total DNA, and the seedlings were identified by PCR with specific primers 3301-F/3301-R, and plants with a band of about 200bp could be successfully amplified, and the PCR products were further sequenced. The results were compared with the target sequence, and the plants with the same sequence were identified as positive transgenic plants.

The obtained transgenic plants were compared with the wild type in terms of growth, and there was no obvious difference in their appearance. The leaves of the plants were expanded, the leaves were emerald green, and the stems were straight. The height of the seedlings reached about 1.5m at the age of 8 months, and they bloomed at the age of 10 months. solid. Samples were collected at the age of 8 months for phenotypic detection as in Example 4, and the obtained detection results were similar to those in Example 4, indicating that the pCAMBIA3301-S2 recombinant vector in the present invention was transformed into plants and the results obtained by the host infecting the plants were consistent, Reproducible.

The above descriptions are only the preferred embodiments of the present invention, and it should be noted that the above preferred embodiments should not be regarded as limitations of the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For those skilled in the art, some improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

sequence listing

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Claims (9)

1. An RNAi fragment, characterized in that it is composed of a modified DNA sequence derived from the CAD gene of Eucalyptus urophylla in a tail-to-tail connection manner; the improved DNA sequence has a DNA sequence shown as SEQ ID NO. 1.

2. The RNAi fragment of claim 1, wherein the DNA sequence of the RNAi fragment is represented in SEQ id No. 2.

3. A recombinant plant expression vector characterized by: comprising the RNAi fragment of claim 1 or 2.

4. The recombinant plant expression vector of claim 3, wherein the recombinant plant expression vector is pCAMBIA3301-S2, which is a recombinant vector constructed based on pCAMBIA 3301.

5. A host cell, characterized in that: comprising an RNAi fragment as defined in claim 1 or 2 or comprising a recombinant plant expression vector as defined in claim 3 or 4.

6. The host cell of claim 5, wherein: the host cell is escherichia coli, agrobacterium or plant cell.

7. Use of an RNAi fragment for modulating lignin synthesis, wherein the RNAi fragment of claim 1 or 2, the recombinant plant expression vector of claim 3 or 4, or the host cell of claim 5 or 6 is used to reduce lignin synthesis in a plant.

8. The method of claim 7, wherein the RNAi fragment or the recombinant plant expression vector is transformed into a plant to direct the reduction of lignin synthesis in the plant.

9. The method of using the RNAi fragment of claim 7 for modulating lignin synthesis, wherein the host cell is infected into a plant.

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