Sugarcane lignin-related gene ScDIR11 and its application in sugarcane breeding for resistance to smut

Abstract

The present invention discloses a sugarcane lignin-related gene ScDIR11 and its application in smut-resistant sugarcane breeding. The inventors found through studying the sugarcane lignin synthesis-related gene ScDIR11 that overexpressing the ScDIR11 gene of sugarcane itself can lead to an increase in the lignin content of transgenic sugarcane, and the transgenic sugarcane infected with sugarcane whip smut showed that its smut incidence rate was significantly reduced, and the content of sugarcane whip smut in the transgenic plants decreased. Based on this, the inventors established a method for obtaining highly smut-resistant sugarcane, and a plasmid containing the sugarcane ScDIR11 gene was genetically transformed into sugarcane through Agrobacterium tumefaciens-mediated method to obtain highly smut-resistant sugarcane materials. Therefore, the present invention is conducive to clarifying the disease resistance mechanism of sugarcane to sugarcane whip smut by cloning the gene ScDIR11, and is applied to the directional molecular breeding of highly disease-resistant sugarcane through genetic engineering means, providing technical support for the use of the sugarcane ScDIR11 gene to prevent and control sugarcane smut.

Description

Technical Field

[0001] The invention belongs to the technical field of sugarcane gene breeding, and in particular relates to a sugarcane lignin-related gene ScDIR11 and application thereof in smut-resistant sugarcane breeding. Background Art

[0002] Sugarcane (Saccharum officinarum L.) is an annual or perennial herb and the world's main sugar and bioenergy crop. Currently, there are hundreds of countries growing sugarcane, among which Brazil has the largest planting area, followed by India and China. Sugarcane production uses stem nodes as seedlings, but sugarcane breeding mainly uses sexual hybridization. Modern sugarcane is an allogeneic high-ploidy aneuploid crop with an extremely complex genetic background. It is very difficult to aggregate many excellent traits into one sugarcane variety. However, sugarcane smut caused by the invasion of sugarcane whip smut fungus into sugarcane has been found in almost all countries and regions where sugarcane is grown. Most of my country's sugarcane is planted in dry land, which is conducive to the overwintering and spread of the winter spores of sugarcane whip smut fungus. In addition, the main varieties grown in China's sugarcane areas are relatively single, and many sugarcane varieties with good agronomic traits, wide adaptability, high yield and high sugar content are susceptible to smut. Therefore, sugarcane smut has become the most serious fungal disease in sugarcane production.

[0003] Sugarcane whip smut is a pathogenic fungus that causes sugarcane smut. It belongs to the Ustilagogueles of the Basidiomycetes. After its binucleate hyphae invade the sugarcane tissue, it continuously absorbs the host's nutrients to grow. In the late stage of infection, a "black whip-like" structure formed by the spores of the sugarcane whip smut wrapped in the sugarcane tissue will grow at the tip of the sugarcane. The structure contains mature dark brown spores and residual plant tissues, and the diseased plants completely lose their economic value. The smut incidence rate of newly planted sugarcane is between 2% and 5%, the incidence rate of one-year perennial roots increases to 8%-15%, and the incidence rate of two-year perennial roots is as high as more than 20%, resulting in an average perennial root period of only two years for sugarcane, which poses a serious threat to the development of my country's sugarcane industry.

[0004] Breeding new disease-resistant varieties is the most economical and effective measure to prevent and control sugarcane smut. Sugarcane is an asexually propagated crop with an extremely complex genetic background. The improvement of sugarcane varieties through hybrid breeding is restricted by many factors. Therefore, breeding excellent sugarcane varieties through genetic engineering can greatly shorten the breeding cycle and avoid the difficulties of hybrid breeding caused by too many repetitive sequences in the sugarcane genome. So far, there is no stable and effective genetic engineering technology to improve sugarcane smut resistance.

[0005] The function of sugarcane DIR gene (ScDIR) has not been systematically elucidated, and there is no report on using sugarcane DIR gene for sugarcane disease resistance breeding. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a sugarcane lignin-related gene ScDIR11 and application thereof in smut-resistant sugarcane breeding.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] The sugarcane ScDIR11 gene has the base sequence of SEQ.ID.NO.1 in the sequence listing.

[0009] A genetic transformation plasmid containing the above-mentioned sugarcane ScDIR11 gene.

[0010] The method for constructing the above genetic transformation plasmid uses the strong promoter Ubi of monocotyledonous plants as the promoter of the genetic transformation vector, connects the Ubi fragment and the pCAMBIA3300-NOS vector with restriction endonucleases Bam HI and Hind III to obtain the plasmid pCAMBIA3300-Ubi-NOS, and connects the sugarcane ScDIR11 gene amplified by PCR with the pCAMBIA3300-Ubi-NOS plasmid to obtain pCAMBIA3300-Ubi-ScDIR11-NOS.

[0011] Application of sugarcane ScDIR11 gene in regulating sugarcane lignin production.

[0012] Application of sugarcane ScDIR11 gene in controlling sugarcane smut.

[0013] Application of sugarcane ScDIR11 gene in sugarcane breeding for resistance to smut.

[0014] The method for obtaining sugarcane with high resistance to smut disease is to genetically transform the above genetic transformation plasmid into sugarcane through Agrobacterium tumefaciens-mediated method.

[0015] In view of the existing problems in the prevention and control of sugarcane smut, the inventors studied the sugarcane ScDIR11 gene and found that overexpression of the ScDIR11 gene of sugarcane itself can lead to an increase in the lignin content of transgenic sugarcane, and the infection of transgenic sugarcane with sugarcane whip smut showed that its smut incidence rate was significantly reduced, and the content of sugarcane whip smut in transgenic plants was reduced. Based on this, the inventors established a method for obtaining highly resistant smut sugarcane, and a plasmid containing the sugarcane ScDIR11 gene was genetically transformed into sugarcane by Agrobacterium tumefaciens-mediated method to obtain highly resistant smut sugarcane materials, thereby improving the resistance of sugarcane to sugarcane whip smut. Therefore, the present invention is conducive to clarifying the disease resistance mechanism of sugarcane to sugarcane whip smut by cloning the lignin synthesis gene ScDIR11 in sugarcane, and is applied to the directional molecular breeding of highly disease-resistant sugarcane through genetic engineering methods, providing technical support for the use of sugarcane ScDIR11 gene to prevent and control sugarcane smut. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the electrophoresis diagram of the PCR amplification product of the ScDIR11 gene.

[0017] Figure 2 This is a schematic diagram of the construction of the genetic transformation vector pCAMBIA3300-Ubi-ScDIR11-NOS.

[0018] Figure 3 This is the electrophoresis diagram of the pCAMBIA3300-Ubi-NOS vector verified by enzyme digestion. In the figure: the small fragment in lane 1 is the Ubi fragment, and the large fragment is the pCAMBIA3300-NOS fragment.

[0019] Figure 4 This is the electrophoresis diagram of the pCAMBIA3300-Ubi-ScDIR11-NOS vector verified by enzyme digestion. In the figure: the small fragment in lane 1 is the ScDIR11 fragment, and the large fragment is the pCAMBIA3300-Ubi-NOS fragment.

[0020] Figure 5 This is the electrophoresis diagram of the Bar gene in the transgenic sugarcane line transformed with the pCAMBIA3300-Ubi-NOS empty plasmid verified by PCR. In the figure: M is a marker, and 1-42 are samples of the transgenic sugarcane lines.

[0021] Figure 6 This is the electrophoresis diagram of transgenic sugarcane transformed with pCAMBIA3300-Ubi-ScDIR11-NOS plasmid verified by PCR. In the figure: A and B verify the Bar gene, C and D verify the ScDIR11-NOS fragment, M is a marker, and 1-39 are transgenic sugarcane line samples.

[0022] Figure 7 This is a heat map of the relative expression of transgenic sugarcane genes transferred into the pCAMBIA3300-Ubi-ScDIR11-NOS plasmid verified by qRT-PCR (Heml 1.0.3.7 software), in which WT is non-transgenic sugarcane, and lines 1-36 are transgenic sugarcane line samples. The color scale of the heat map represents the expression level, blue indicates low transcript abundance level, and red indicates high transcript abundance level.

[0023] Figure 8 This is a graph analyzing the lignin content of sugarcane. In the graph: the ordinate represents the lignin content per milligram of sugarcane sprouts, the abscissa represents the sugarcane plants, WT represents non-transgenic sugarcane, 35S::00 represents the sugarcane line introduced with the pCAMBIA3300-Ubi-NOS vector, and 35S::ScDIR11 represents the transgenic sugarcane line introduced with the ScDIR11 gene.

[0024] Fig. 9 This is an analysis chart of the smut incidence of sugarcane inoculated with Ustilago officinalis. In the figure: the ordinate represents the smut incidence of sugarcane, the abscissa represents the statistical time of the onset of each sugarcane line, n is the sugarcane plant, WT is non-transgenic sugarcane, 35S::00 represents the sugarcane line transformed with the pCAMBIA3300-Ubi-NOS vector, and 35S::ScDIR11 represents the transgenic sugarcane line transformed with the ScDIR11 gene.

[0025] Fig.10 This is a graph analyzing the smut content of sugarcane, in which: the ordinate represents the content of sugarcane whip smut, which is expressed as the number of copies of specific fragments per microgram of sugarcane DNA, with 10 as the base for the logarithm of the gene copy number; the abscissa represents the sugarcane strain, WT is non-transgenic sugarcane, 35S::00 represents the sugarcane strain transformed with the pCAMBIA3300-Ubi-NOS vector, and 35S::ScDIR11 represents the transgenic sugarcane strain transformed with the ScDIR11 gene. DETAILED DESCRIPTION

[0026] Example 1 Cloning of the full-length coding region of the sugarcane ScDIR11 gene

[0027] (1) Take 0.3 g of fresh sugarcane sprouts and extract total RNA according to the instructions of Transgen TransZol Plant Kit from Quanshijin Biotechnology Co., Ltd., and then use the RNA of Novozyme Biotechnology Co., Ltd. II 1st Strand cDNA Synthesis Kit was used to reverse transcribe RNA into cDNA and stored at -80℃ for later use.

[0028] (2) Download the sequence of ScDIR11 gene from the sugarcane genome database, and use SnapGene software to design primers containing Bam HI and SacI at both ends to clone the ScDIR11 gene. The primer sequence is:

[0029] Forward primer 5′-CGCGGATCCATGGCCAAAAGCAAGCTTAGTACC-3′

[0030] Reverse primer 5′-CGCGAGCTCCTACACGCGCAGGTGCA-3′

[0031] (3) The cDNA fragment of the ScDIR11 gene was amplified by PCR based on the obtained cDNA template.

[0032] (4) The reaction system is as follows: add 100 ng of cDNA as template, 10 μL of Prime STAR Max Premix, 1 μL of forward and reverse primers, and finally ddHO.2 O to make up to 20μL; PCR reaction program: 95℃ pre-denaturation for 5min, 95℃ denaturation for 15sec, 60℃ annealing for 20sec, 72℃ extension for 30sec, 30 cycles, 72℃ extension for another 2min.

[0033] (5) PCR products were detected by agarose gel electrophoresis. Figure 1 As shown, a 552 bp ScDIR11 gene fragment was obtained.

[0034] Example 2 Construction of genetic transformation plasmid pCAMBIA3300-Ubi-ScDIR11-NOS

[0035] The construction process is as follows Figure 2 As shown, please proceed as follows:

[0036] (1) The Ubi fragment and the pCAMBIA3300-NOS vector were connected with restriction endonucleases Bam HI and Hind III to obtain the plasmid pCAMBIA3300-Ubi-NOS. The results of enzyme digestion verification were as follows: Figure 3 shown.

[0037] (2) The ScDIR11 gene amplified by PCR was connected to the pCAMBIA3300-Ubi-NOS plasmid to obtain the genetic transformation vector pCAMBIA3300-Ubi-ScDIR11-NOS. The results of enzyme digestion verification were as follows: Figure 4 shown.

[0038] (3) The recombinant vector was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and the correctly sequenced pCAMBIA3300-Ubi-ScDIR11-NOS vector was stored at -80°C for future use.

[0039] Example 3 Genetic transformation of sugarcane with ScDIR11 gene

[0040] (1) The pCAMBIA3300-Ubi-NOS empty plasmid and the pCAMBIA3300-Ubi-ScDIR11-NOS genetic transformation plasmid were introduced into sugarcane embryonic callus (sugarcane variety ROC 22 susceptible to smut) by Agrobacterium-mediated method. The Bar gene was the selection marker gene on the pCAMBIA3300 plasmid.

[0041] (2) Identification of genetically modified sugarcane

[0042] PCR and qRT-PCR tests showed that the DNA of a transgenic sugarcane line contained the Bar gene ( Figure 5); the expression level of ScDIR11 gene in the DNA of another strain was significantly increased, indicating that the ScDIR11 gene was successfully overexpressed in sugarcane ( Figure 6 and Figure 7 ).

[0043] Example 4 Detection of lignin content in sugarcane

[0044] The buds of transgenic sugarcane and non-transgenic sugarcane were taken and dried at 80℃ for two days, and the lignin content was determined using the lignin detection kit of Beijing Solebaugh Technology Co., Ltd. Figure 8 As shown, the lignin content of transgenic sugarcane was significantly higher than that of non-transgenic sugarcane and empty-transplanted sugarcane, again indicating that the ScDIR11 gene was successfully overexpressed in the transgenic sugarcane line.

[0045] Example 5 Testing the resistance of sugarcane to Ustilago canefolia

[0046] (1) The sugarcane whip smut fungus haploids JG35 and JG36 were inoculated into liquid YEPS medium and cultured at 28°C and 200 rpm in a shaking incubator until OD 600 The absorbance value at that time was 1.0. Collect the bacteria and resuspend them in sterile water of equal volume to the bacterial solution to form the infection solution.

[0047] (2) Clean the rooted sugarcane tissue culture seedlings, put them into the dye solution, soak the roots, and culture them in a 28°C light culture room for 3 days. After soaking, plant them on the seedling medium and culture them in an artificial climate room with a light / dark ratio of 16h / 8h, a temperature of 28°C, and a humidity of 80% to count the incidence of sugarcane smut.

[0048] (3) Fig. 9 As shown, on the 41st day after inoculation, the diseased plants first appeared in the non-transgenic sugarcane; on the 46th day after infection, the diseased plants appeared in the 35S::00 sugarcane line; on the 61st day, the diseased plants appeared in the two 35S::ScDIR11 sugarcane lines. 150 days after the inoculation of sugarcane whip smut fungus, the disease incidence rates of the non-transgenic sugarcane plants and the 35S::00 line were 37 / 50 (74.0%) and 36 / 50 (72.0%), respectively, and the disease incidence rate of the 35S::ScDIR11 sugarcane line was 4 / 35 (11.4%). The sugarcane plants in the control group soaked in sterile water did not get sick during the statistical time.

[0049] Example 6 Detection of the Ustilago content at the tip of the sugarcane stem after inoculation with Ustilago cane

[0050] (1) 150 days after the sugarcane whip smut was inoculated on the sugarcane seedlings, the stem tip growth point of the sugarcane was taken to extract the total DNA of the sugarcane. The DNA was used as a template and specific primers bE4 and bE8 were used to detect the sugarcane whip smut. The primer sequences are:

[0051] Primer bE4 5′-CGCTCTGGTTCATCAACG-3′

[0052] Primer bE8 5′-TGCTGTCGATGGAAGGTGT-3′

[0053] (2) Fig.10 As shown, the disease-free sugarcane overexpressing the ScDIR11 gene also expressed the sugarcane whip smut fungus-specific gene fragment, but its expression level was significantly lower than that of the sugarcane without overexpressing the ScDIR11 gene, indicating that the overexpression of the ScDIR11 gene inhibited the proliferation of the sugarcane whip smut fungus.

Claims

1. Application of sugarcane ScDIR11 gene in controlling sugarcane smut.

2. Application of sugarcane ScDIR11 gene in sugarcane breeding with resistance to smut.

3. A method for obtaining sugarcane with high resistance to smut, characterized in that: The genetic transformation plasmid containing the sugarcane ScDIR11 gene was genetically transformed into sugarcane by Agrobacterium tumefaciens-mediated method.

4. The method for obtaining highly resistant sugarcane to smut according to claim 3, characterized in that: The sugarcane ScDIR11 gene is the base sequence of SEQ.ID.NO.1 in the sequence list.

5. The method for obtaining sugarcane with high resistance to smut according to claim 4, characterized in that: The genetic transformation plasmid is constructed according to the following method: using the strong promoter Ubi of monocotyledonous plants as the promoter of the genetic transformation vector, connecting the Ubi fragment and the pCAMBIA3300-NOS vector with restriction endonucleases BamHI and Hind III to obtain the plasmid pCAMBIA3300-Ubi-NOS, and connecting the sugarcane ScDIR11 gene amplified by PCR with the pCAMBIA3300-Ubi-NOS plasmid to obtain pCAMBIA3300-Ubi-ScDIR11-NOS.

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