阅读说明：本技术 通过超表达秋茄KoCSD3提高植物镉耐受性的方法 (Method for improving cadmium tolerance of plants by over-expressing Kandelia candel KoCSD3 ) 是由 潘铖烺 陈建明 严重玲 于 2020-06-16 设计创作，主要内容包括：本发明属于基因表达技术领域,公开了一种通过超表达秋茄KoCSD3提高植物镉耐受性的方法,通过超表达秋茄KoCSD3提高植物镉耐受性的方法使用表达载体pCAMBIA1302-KoCSD 3；以pCAMBIA1302载体为骨架,利用NcoI和BstEII两个酶切位点,将原有的GUS基因片段切除；再利用无缝克隆技术将带有双酶切位点的KoCSD3基因片段分别连入载体中,构建表达载体。超表达KoCSD3的烟草较野生型表现出更高的镉耐受性,表现在植株的根系生长发育情况上,也发现KoCSD3可提高烟草根系木质素含量且促进维管组织的木质化。与超表达秋茄其他SOD成员相比,超表达KoCSD3可显著降低烟草中镉的累积。(The invention belongs to the technical field of gene expression, and discloses a method for improving the cadmium tolerance of plants by over-expressing Kandelia candel KoCSD3, which uses an expression vector pCAMBIA1302-KoCSD3 by over-expressing Kandelia candel KoCSD3 to improve the cadmium tolerance of plants; taking a pCAMBIA1302 vector as a framework, and cutting an original GUS gene fragment by utilizing two enzyme cutting sites of NcoI and BstEII; and respectively connecting the KoCSD3 gene segments with double enzyme cutting sites into a vector by utilizing a seamless cloning technology to construct an expression vector. Tobacco over-expressing KoCSD3 showed higher cadmium tolerance than wild-type, as demonstrated by the growth and development of the root system of the plants, and KoCSD3 was also found to increase tobacco root system lignin content and promote lignification of vascular tissue. Over-expression of KoCSD3 significantly reduced the accumulation of cadmium in tobacco compared to over-expression of other members of kandelia candel SOD.)

1. A method for improving the cadmium tolerance of plants by over-expressing Kandelia candel KoCSD3 is characterized in that an expression vector pCAMBIA1302-KoCSD3 is used in the method for improving the cadmium tolerance of plants by over-expressing Kandelia candel KoCSD 3.

2. A method of constructing the pCAMBIA1302-KoCSD3 expression vector of claim 1, wherein the method of constructing the pCAMBIA1302-KoCSD3 expression vector comprises the steps of:

firstly, taking a pCAMBIA1302 vector as a skeleton, and cutting an original GUS gene fragment by utilizing two enzyme cutting sites of NcoI and BstEII;

secondly, respectively connecting the KoCSD3 gene segments with double enzyme cutting sites into a vector by utilizing a seamless cloning technology to construct an expression vector pCAMBIA1302-KoCSD 3.

3. The method of claim 2, wherein the method of constructing the pCAMBIA1302-KoCSD3 expression vector uses the primer sequences: SEQ ID NO: 1.

Technical Field

The invention belongs to the technical field of gene expression, and particularly relates to a method for improving cadmium tolerance of plants by over-expressing Kandelia candel KoCSD 3.

Background

At present, cadmium (Cd) is one of common environmental pollutants with high toxicity, has the characteristics of strong mobility, difficult degradation and the like, is easily absorbed by plant root systems, transported to overground parts of plants and accumulated in plant tissues and organs (Benavides, et al.2005; Gill, et al.2012). Soil investigation by Zhang et al in 11 provinces of China revealed that at least 13330 hectares of farmland in China suffered from Cd pollution (Zhang, et al.2000). When the content of cadmium in the plant body exceeds a certain amount (5-10 mu g/g dry weight), toxic effects can be generated on the plant, such as changes of plant morphology, physiology and biochemistry and cellular molecular level, and irreversible damage can be caused on all aspects of normal growth and development of the plant. It is noteworthy that plants are the main source of exposure to cadmium in humans, which accumulates in different organs of plants and enters the human body through the food chain, posing a great threat to human health (Rainbow, et al.2011; satarurg, et al.2002).

As a typical salt-tolerant mangrove plant, Kandelia obovata (Kandelia obovata) is the dominant species of mangrove forest in the southeast of China, and has strong tolerance to complex environments with high organic matter, low oxygen and various heavy metal pollution in staggered land and sea (Weng, et al.2012). The scholars in the early years think that the endothelial layer of the root system of the salt-repellent mangrove plant has an ultrafiltration mechanism (Tolinson P.B.1986), which can prevent heavy metals from entering the phloem and further reduce the transport of the heavy metals to the overground part. Further research results in Junyi (2014) show that the secondary thickening phenomenon of the outer cortex of the root system of Kandelia has the effect similar to an ultrafiltration mechanism, and the secondary thickening phenomenon of the outer cortex precedes the formation of the Kirschner tape of the inner cortex of the root in the cadmium stress process and is considered as a 'cadmium migration barrier' before the formation of the Kirschner tape. Recently, researchers found that this impermeable root surface barrier also regulates the flux of rhizosphere gas, moisture and solute, can effectively block the migration of various heavy metals such as cadmium, lead, zinc and copper into roots, and is the key to ensure the normal growth of Kandelia candel in the heavy metal enriched wetland environment (Pi, et al.2009, Cheng, et al.2014). In the early cadmium stress experiment, the inventor finds that Kandelia candel Cu/ZnSOD3(KoCSD3) plays an important role in the cadmium tolerance process of the root system of Kandelia candel (Pan, et al.2020). However, the current research on the mangrove plant Kandelia candel SOD still relates to the enzyme activity and the antioxidant effect, and the specific function of the Kandelia candel SOD in the aspect of cadmium tolerance is not clear. Therefore, the method for improving the heavy metal tolerance of the plant by exogenously over-expressing the Kandelia candel SOD gene by using the genetic engineering technology has wide application prospect.

Through the above analysis, the problems and defects of the prior art are as follows: at present, the research on the mangrove plant Kandelia candel SOD is still related to the enzyme activity and the antioxidant effect, and the specific function of the Kandelia candel SOD in the aspect of cadmium tolerance is not clear.

The difficulty in solving the above problems and defects is: as the Kandelia candel is a non-model plant and the plant is rich in secondary metabolites such as tannin, the difficulty in-situ research on the gene function is high, so that most people do not fully know the biological function of the Kandelia candel SOD.

The significance of solving the problems and the defects is as follows: the method for improving the heavy metal tolerance of the plant by exogenously over-expressing the Kandelia candel SOD gene by using the genetic engineering technology has wide application prospect.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for improving the cadmium tolerance of plants by over-expressing Kandelia candel KoCSD 3.

The invention is realized by a method for improving the cadmium tolerance of plants by over-expressing Kandelia candel KoCSD3, and the method for improving the cadmium tolerance of plants by over-expressing Kandelia candel KoCSD3 uses an expression vector pCAMBIA1302-KoCSD 3. Overexpression is a method, namely the gene of Kandelia candel is transformed into other plants, and the gene is found to promote the accumulation of cadmium in a mode of promoting vascular tissue thickening.

The invention also aims to provide a construction method of the pCAMBIA1302-KoCSD3 expression vector, and the construction method of the pCAMBIA1302-KoCSD3 expression vector comprises the following steps:

firstly, taking a pCAMBIA1302 vector as a skeleton, and cutting an original GUS gene fragment by utilizing two enzyme cutting sites of NcoI and BstEII;

secondly, respectively connecting the KoCSD3 gene segments with double enzyme cutting sites into a vector by utilizing a seamless cloning technology to construct an expression vector pCAMBIA1302-KoCSD 3.

Further, the primer sequence used in the construction method of the pCAMBIA1302-KoCSD3 expression vector is as follows: SEQ ID NO: 1.

by combining all the technical schemes, the invention has the advantages and positive effects that: tobacco over-expressing KoCSD3 showed higher cadmium tolerance than wild-type, mainly in the root growth and development of plants (fig. 3), and KoCSD3 was also found to increase tobacco root lignin content and promote lignification of vascular tissue (fig. 4). At the same time, overexpression of KoCSD3 significantly reduced the accumulation of cadmium in tobacco compared to overexpression of other members of kandelia (KoFSD2) (fig. 5).

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.

FIG. 1 is a flow chart of the construction method of the pCAMBIA1302-KoCSD3 expression vector provided by the embodiment of the invention.

FIG. 2 is a schematic diagram of the plasmid map of pCAMBIA1302-KoCSD3 provided by the embodiment of the present invention.

FIG. 3 is a schematic diagram of the growth of Nicotiana benthamiana roots promoted by overexpression of KoCSD3 under cadmium stress according to the embodiment of the present invention;

in fig. 3: (a) the overexpression of KoCSD3 was compared with the growth of wild type Nicotiana benthamiana plants after 30 days of cadmium stress (+ Cd: 200. mu.M; -Cd: 0. mu.M) (scale bar in the figure is 1 cm); (b) the root development conditions of different Benzenbacco under cadmium stress are observed under a dissecting mirror (the surface ruler in the figure is 0.5 cm).

FIG. 4 is a schematic representation of the enhancement of tobacco root lignin accumulation by over-expression of KoCSD3 provided by an embodiment of the present invention;

in fig. 4: (A) measuring the lignin content of the root systems of the transgenic tobacco and the wild tobacco (growing for 30 days); (B) the 15-day tobacco seedlings (over-expressed KoCSD3 and wild type) were paraffin sectioned and lignin fluorescence was observed using a laser confocal microscope. The degree of lignification of each section was analyzed using Image-pro plus 6.0 (15 sections per line, 3 400-fold fields per section for photographic analysis; 25 μm ruler in the figure).

FIG. 5 is a schematic diagram of the determination of root cadmium accumulation under cadmium stress of tobacco plants over-expressed KoCSD3 and KoFSD2 according to the embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In view of the problems of the prior art, the present invention provides a method for improving cadmium tolerance of plants by over-expressing Kandelia candel KoCSD3, and the present invention is described in detail below with reference to the accompanying drawings.

As shown in FIG. 1, the construction method of the pCAMBIA1302-KoCSD3 expression vector provided by the invention comprises the following steps:

s101: taking a pCAMBIA1302 vector as a framework, and cutting an original GUS gene fragment by utilizing two enzyme cutting sites of NcoI and BstEII;

s102: and respectively connecting the KoCSD3 gene segments with double enzyme cutting sites into a vector by using a seamless cloning technology to construct an expression vector pCAMBIA 1302-KoCSD.

The technical solution of the present invention is further described below with reference to the accompanying drawings.

The invention uses pCAMBIA1302 vector as a framework, and utilizes two enzyme cutting sites of NcoI and BstEII to cut out the original GUS gene fragment. And respectively connecting the KoCSD3 gene segments with double enzyme cutting sites into a vector by using a seamless cloning technology to construct an expression vector pCAMBIA1302-KoCSD 3. The primers for vector construction are shown in Table 1. A schematic of the vector construction is shown in FIG. 2. The recombinant plasmid is transferred into Escherichia coli DH5 alpha for sequencing verification.

TABLE 1 pCAMBIA1302-KoCSD3 vector construction primers (SEQ ID NO: 1)

Tobacco expressing KoCSD3 showed higher cadmium tolerance than wild type, mainly in the root growth and development of plants (fig. 3), and KoCSD3 was found to increase tobacco root lignin content and promote lignification of vascular tissue (fig. 4). At the same time, overexpression of KoCSD3 significantly reduced the accumulation of cadmium in tobacco compared to overexpression of other members of kandelia (KoFSD2) (fig. 5).

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

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