CN109706155B - pOsHEN1::OsSPL14 gene expression cassette and its construction method and application - Google Patents

Abstract

The present invention provides a pOsHEN1::OsSPL14 gene expression cassette in order to balance the opposing aspects of rice stress resistance and development. The pOsHEN1::OsSPL14 gene expression cassette includes an OsHEN1 promoter and an OsSPL14 gene, and the OsSPL14 gene is connected to the OsHEN1 promoter. Downstream, the present invention also provides a recombinant expression vector containing the aforementioned pOsHEN1::OsSPL14 gene expression cassette, and provides the aforementioned construction method of the recombinant expression vector. The gene expression cassette and recombinant expression vector of the present invention can produce excellent yield traits in rice, such as increased stem diameter and single tiller yield and/or improved rice resistance to Bacterial blight PXO99A. When the transgenic line was not invaded by PXO99A, the expression of OsSPL14 in the background of transgenic rice was slightly increased, and the characteristics of OsSPL14 in regulating the ideal plant type of rice were exerted, and tillering was appropriately reduced to increase the effective tiller number, increase stem diameter, lodging resistance and improve Single tiller yield of rice. When the transgenic line was invaded by PXO99A, the transcription level of the downstream OsSPL14 gene was greatly increased, which could specifically enhance the resistance to bacterial blight.

Description

pOsHEN1::OsSPL14 gene expression cassette and its construction method and application

technical field

The invention relates to the technical field of plant genetic engineering, in particular to the construction and breeding application of a pOsHEN1::OsSPL14 gene expression cassette.

Background technique

Xanthomonas oryzae pv.oryzae, Xoo are pathogenic bacteria that cause bacterial blight in rice. Rice bacterial blight occurs in all rice regions in my country and is the main disease of rice. Under high temperature, the seedling disease spots are short stripes, small and narrow. After expansion, the leaves quickly wither, yellow and wither. There are many grains and broken rice. harvest, which has a greater impact on yield. Therefore, it is very necessary to improve rice resistance to bacterial blight through genetic engineering.

Plants, including crops, are mostly fixed in the growing place, and they can change the signal network of endogenous growth and development according to the constantly changing external environment, so as to better adapt to the external environment. When infected by pathogenic bacteria, plants will adjust energy from growth and development to defense signaling network, so the process of disease resistance often manifests as inhibition of growth and development. In order to improve the disease resistance of crops, related research in the laboratory screened a group of mutants that constitutively activate the resistance response. These mutants often show cell death, high expression of disease resistance-related genes, and high accumulation of resistant substances, which can ultimately effectively inhibit the reproduction of bacteria or fungi. However, most of these mutants have dwarf plants and reduced fertility in terms of growth and development. , the decline of seed yield and other disadvantages. The study of these disease resistance genes has greatly contributed to understanding the molecular pathways of plant immunity, but the application of these genes in genetic breeding production is hampered by the often accompanying developmental defects mentioned above.

Therefore, in improving the resistance of rice to bacterial blight, it is necessary to balance the opposing sides of rice stress resistance and development. It has important and broad application value to develop a transgenic material that can not only specifically increase rice disease resistance, but also increase rice yield.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a gene expression cassette that can not only specifically increase the resistance to bacterial blight of rice, but also increase the yield of rice, and its construction method and application, in order to address the deficiencies of the prior art.

The technical scheme of the present invention is as follows:

The first object of the present invention is to provide a pOsHEN1::OsSPL14 gene expression cassette, the pOsHEN1::OsSPL14 gene expression cassette includes an OsHEN1 promoter and an OsSPL14 gene, and the OsSPL14 gene is connected downstream of the OsHEN1 promoter.

The nucleotide sequence of the OsHEN1 promoter is shown in SEQ ID No.5, and the nucleotide sequence of the OsSPL14 gene is shown in SEQ ID No.6.

The second object of the present invention is to provide a recombinant expression vector containing the aforementioned pOsHEN1::OsSPL14 gene expression cassette.

Further, the recombinant expression vector is obtained by inserting the pOsHEN1::OsSPL14 gene expression cassette of claim 1 between the EcoRI and HindIII sites of the pCAMBIA1305.1 plasmid vector.

The application of the aforementioned pOsHEN1::OsSPL14 gene expression cassette or the aforementioned recombinant expression vector in improving rice crops, the improvement comprising: improving the excellent yield traits of rice and/or improving the resistance of rice to Bacterial blight PXO99A.

Further, the excellent yield traits of rice include: reducing tillers to increase the number of effective tillers and reducing production consumption, and/or increasing stem diameter to enhance lodging resistance, and/or increasing yield per ear of rice to increase rice yield

The third object of the present invention is to provide the construction method of the aforementioned recombinant expression vector, and the construction method comprises the following steps:

S1: Using rice Nipponbare DNA as template, using OsHEN1 promoter-specific primer pOsHEN1-F:

tatgaccatgattacgaattcTTATGTGCACTAGAAACTATCTGAGGAC (SEQ ID No. 1) and pOsHEN1-R: CAAACGCCCAAAAAAAACAA (SEQ ID No. 2) were amplified by PCR, and the amplified product of the promoter of OsHEN1 was cloned;

S2: Using rice Nipponbare DNA as a template, using OsSPL14 gene-specific primer OsSPL14-F:

ttgttttttttgggcgtttgTTCCGTCTCTTTCCTCTCTCTTCT (SEQ ID No. 3) and OsSPL14-R:

tggtctttgtagtcaagcttCAGAGACCAATCCATCGTGTTG (SEQ ID No. 4) was amplified by PCR, and the genomic DNA amplification product of OsSPL14 was cloned;

S3: The amplified fragments obtained from S1 and S2 were connected to the plasmid vector of pCAMBIA1305.1 double-digested by EcoRI and HindIII by homologous recombination method to obtain a recombinant vector containing the pOsHEN1::OsSPL14 expression cassette.

Further, the specific operation steps of S3 are as follows: using the homologous recombination kit with the product number C113-02 of Nanjing Novizan Company, the promoter fragment of OsHEN1, the genomic DNA fragment of OsSPL14 and pCAMBIA1305 after double digestion with EcoRI and HindIII were used. 1. The linear fragments were connected together, and after E. coli transformation, a recombinant vector containing the pOsHEN1::OsSPL14 expression cassette was obtained by monoclonal identification.

The homologous recombination system is as follows:

The recombination system was configured according to the above system, mixed with a pipette, placed at 37°C for 30 minutes, and then placed on ice for 5 minutes to carry out the next step of Escherichia coli transformation.

Transform E. coli

1. Take out a tube (100 μl) of competent bacteria from the -70°C ultra-low temperature freezer, and melt the competent bacteria in an ice bath for 5-10 minutes.

2. Add 5 μl of the ligated recombinant plasmid, shake gently and place on ice for 20 min.

3. Shake gently, insert it into a 42°C water bath for 90 seconds to heat shock, then quickly put it back on ice and let it stand for 2 minutes.

4. Add 500 μl of anti-LB medium to the above tubes in the ultra-clean workbench and mix gently, and then fix to the shaker.

Shake at 37°C for 45min on the spring frame of the bed.

5. Take the above transformation mixture on the ultra-clean workbench and pour it into a solid LB plate culture dish containing kanamycin antibiotics.

Coat with a glass coating rod burned by an alcohol lamp, and dry the surface of the medium in an ultra-clean bench.

6. Invert the medium and put it into a 37°C constant temperature incubator overnight.

7. Pick a single clone colony for colony PCR identification, sequence and then identify the positive plasmid pOsHEN1::OsSPL14 vector.

The pOsHEN1::OsSPL14 gene expression cassette of the present invention or the recombinant expression vector containing the pOsHEN1::OsSPL14 gene expression cassette can improve rice excellent yield traits such as stem diameter and single ear yield to increase rice yield and/or improve rice resistance to rice bacterial blight The mechanism of action of the resistance of pathogen PXO99A is as follows:

Xanthomonas oryzae pv. oryzae (Xoo) secretes different effectors directly into rice cells through the Typethree secretion system (T3SS), and these effectors bind to interacting targets in rice for produce a disease-resistant or susceptible response. Transcription activation-like (TAL) effector, as the most important tritype effector of Xanthomonas oryzae, plays a key role in its interaction with rice. The rice gene OsHEN1 is directly regulated by the Tal9A transcriptional activation effector of Xoo PXO99A. The Tal9A transcriptional activation effector of Xoo PXO99A can specifically bind to the Tal9A effector binding site of Xoo PXO99A at the promoter-25bp of OsHEN1, and activate the Tal9A effector binding site of Xoo PXO99A. OsHEN1 promoter.

Rice OsSPL14 (IPA1) is an ideal plant type gene of rice. Overexpression of OsSPL14 reduces rice tiller number. Fine regulation (up-regulation of expression) of OsSPL14 expression can increase rice yield by reducing total rice tiller number and increasing rice effective tiller number. .

In addition, the inventors unexpectedly discovered that OsSPL14 also plays an important role in the disease resistance of rice for the first time. In rice, up-regulation of OsSPL14 gene expression can also significantly enhance resistance to bacterial blight.

The pOsHEN1::OsSPL14 gene expression cassette constructed in the present invention is connected to the OsSPL14 gene downstream of the inducible promoter OsHEN1, and after rice is transformed, a rice transgenic line containing the pOsHEN1::OsSPL14 gene expression cassette is obtained.

The OsHEN1 promoter mainly contains the target site of the PXO99A pathogenic factor. At the same time, the OsSPL14 gene sequence of most rice materials is the same, and the sequence differences between individual materials have the same function. Therefore, the technical solution of the present invention can be applied to various rice materials. Variety.

When the transgenic line is not invaded by PXO99A, the presence of OsSPL14 in the expression cassette slightly increases the expression of OsSPL14 in the background of transgenic rice. In this state, it can play the role of OsSPL14 in regulating rice tillering, which can appropriately reduce tillering. In order to increase the number of effective tillers, reduce production consumption, increase stem diameter, increase lodging resistance and improve single ear yield of rice, and can increase rice yield through reasonable dense planting.

When the transgenic line was invaded by PXO99A, the PXO99A tal9A transcriptional activation effector could specifically bind to the Tal9A effector binding site of Xoo PXO99A at -25 bp on the OsHEN1 promoter, and the OsHEN1 promoter was induced by Tal9A of XooPXO99A. Specific activation, thereby greatly increasing the transcription level of the downstream OsSPL14 gene, to achieve specific enhancement of the disease resistance to PXO99A bacterial blight under the infection of exogenous bacterial blight PXO99A.

At the same time, the OsHEN1 promoter is an inducible promoter. Due to the spatiotemporal specificity of the inducible promoter, the target OsSPL14 gene can be overexpressed only in the part infected by PXO99A in plants, and the expression level in other organs and tissues is not affected. Therefore, the OsSPL14 gene expression in the infected part is greatly up-regulated (tens to hundreds of times of increase), effectively enhancing the target resistance, while the OsSPL14 gene expression in other organs and tissues is not affected, avoiding the overexpression of OsSPL14 in the whole plant , resulting in excessive reduction of tiller numbers and severe yield reduction. While effectively resisting diseases, it does not affect the yield of rice, thereby obtaining an excellent rice variety that can increase the yield of rice and can enhance the disease resistance to the bacterial blight of rice PXO99A.

The beneficial effects achieved by the technical solution of the present invention are:

After the pOsHEN1::OsSPL14 gene expression cassette constructed in the present invention is transformed into rice, when the transgenic line is not invaded by PXO99A, the tiller is appropriately reduced, so as to ensure the effective tiller number, reduce production consumption, increase stem diameter and lodging resistance and improve rice Yield per ear, and can be increased by reasonable dense planting. When the transgenic line is invaded by PXO99A, it can specifically enhance the resistance to bacterial blight PXO99A in specific parts of rice, and it will not affect the overall OsSPL14 gene expression of the plant, and avoid serious yield reduction due to excessive reduction in the number of tillers. It balances the opposing aspects of rice stress resistance and development, so as to obtain excellent rice varieties that can increase rice yield and enhance disease resistance, and our HS transgenic lines can precisely regulate the expression of OsSPL14 through a variety of breeding methods. , has broad application and market prospects in the agricultural field.

Description of drawings

Figure 1. Schematic diagram of pCAMBIA1305.1 vector plasmid map and multiple cloning site information.

Fig. 2 The pOsHEN1::OsSPL14 gene expression cassette was transformed into rice, and the plant type diagrams of four transgenic lines (HS-2, HS-9, HS-8, Hs-4) were obtained.

Figure 3. The four transgenic lines (HS-2, HS-9, HS-8, Hs-4) had up-regulated background OsSPL14 expression in the uninduced state of PXO99A.

Figure 4. Statistics of plant height and tiller of four transgenic lines (HS-2, HS-9, HS-8, Hs-4) in the state not induced by PXO99A;

In the figure: a is the histogram of plant height comparison of HS-2, HS-9, HS-8, Hs-4 and wild type NIP; b is HS-2, HS-9, HS-8, Hs-4 and wild type NIP tiller comparison histogram; n.d. indicates the significant level of the difference between the transgenic line and the wild-type control at P>0.05; asterisk (*) indicates that the significant level of the difference between the transgenic line and the wild-type control is at P<0.05; Double asterisks (**) indicate the significance level of the difference between the transgenic lines and the wild-type control at P<0.01.

Figure 5. Statistics of the stem diameter of the third node of the four transgenic lines (HS-2, HS-9, HS-8, Hs-4) without induction by PXO99A;

In the figure: a is the comparison of HS-2, HS-9, HS-8, Hs-4 and wild-type NIP third node stem, b is HS-2, HS-9, HS-8, Hs-4 and wild type Type NIP third section stem diameter comparison histogram; asterisk (*) indicates that the significance level of the difference between transgenic lines and wild-type control is at P<0.05; double asterisks (**) indicate that transgenic lines are compared with wild-type control The significance level of the difference compared to the control is at P<0.01.

Figure 6 is a graph comparing the main panicle of four transgenic lines (HS-2, HS-9, HS-8, and Hs-4) with wild-type NIP in the state not induced by PXO99A;

Figure 7. Bar graph comparing four transgenic lines (HS-2, HS-9, HS-8, Hs-4) with wild-type NIP branches in a state not induced by PXO99A;

In the figure: a is the histogram comparing the primary branches of HS-2, HS-9, HS-8, Hs-4 and wild-type NIP; b is the comparison of HS-2, HS-9, HS-8, Hs-4 and The histogram of the secondary branches of wild-type NIP; c is the histogram of the number of spikelets in the main spike of HS-2, HS-9, HS-8, Hs-4 and wild-type NIP; asterisks (*) indicate transgenic lines The significance level of the difference compared with the wild-type control is at P<0.05; the double asterisk (**) indicates that the significance level of the difference between the transgenic line and the wild-type control is at P<0.01.

Fig. 8 Statistics of the number of main panicles of four transgenic lines (HS-2, HS-9, HS-8, Hs-4) without induction by PXO99A;

In the figure: a is the comparison of the number of main spikes of HS-2, HS-9, HS-8, Hs-4 and wild type NIP, b is HS-2, HS-9, HS-8, Hs-4 and wild type Type NIP main panicle yield comparison histogram; asterisk (*) indicates that the significant level of difference between transgenic lines and wild-type control is at P<0.05; double asterisks (**) indicate that transgenic lines are compared with wild-type control The significance level of the difference was at P<0.01.

Figure 9 Fluorescence quantitative PCR identification of the expression of OsSPL14 in three transgenic lines (HS-2, HS-9, HS-8, HS-4) induced by PXO99A;

Figure 10. Statistics of disease resistance of four transgenic lines (HS-2, HS-9, HS-8, Hs-4) in the induced state;

In the figure: a. is the comparison of the lengths of HS-2, HS-9, HS-8, Hs-4 and wild-type NIP lesions, b. is HS-2, HS-9, HS-8, Hs-4 and The histogram of the length of wild-type NIP lesions; c. is the histogram of the proportion of increased resistance of HS-2, HS-9, HS-8, and Hs-4; asterisks (*) indicate the comparison between the transgenic lines and the wild-type control The significance level of the ratio difference is at P<0.05; the double asterisk (**) indicates that the significance level of the difference between the transgenic lines and the wild-type control is at P<0.01.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention. The raw materials involved in the following examples are commercially available unless otherwise specified, and the detection methods involved are conventional methods unless otherwise specified.

Example 1 Construction of pOsHEN1::OsSPL14 gene expression cassette and construction of recombinant expression vector containing pOsHEN1::OsSPL14 gene expression cassette

S1: Design and clone pOsHEN1-F primer at about 2000bp upstream of the transcription start site of rice OsHEN1 gene, design pOsHEN1-R primer at the end of the 5' untranslated region of OsHEN1, use rice Nipponbare DNA as template, and use OsHEN1 promoter-specific primer The primers pOsHEN1-F: tatgaccatgattacgaattcTTATGTGCACTAGAAACTATCTGAGGAC (SEQ ID No. 1) and pOsHen1-R: CAAACGCCCAAAAAAAACAA (SEQ ID No. 2) were amplified by PCR, and the amplified product of the promoter of OsHen1 was cloned;

S2: Design and clone the OsSPL14-F primer based on the transcription start site of OsSPL14, design and clone the OsSPL14-R primer about 500 bp downstream of the OsSPL14 transcription termination site, use the rice Nipponbare DNA as the template, and use the OsSPL14 gene-specific primer OsSPL14-F: ttgtttttttttgggcgtttgTTCCGTCTCTTTCCTCTCTCTTCT (SEQ ID No.3) and OsSPL14-R: tggtctttgtagtcaagcttCAGAGACCAATCCATCGTGTTG (SEQ ID No.4) were amplified by PCR, and the genomic DNA amplification product of OsSPL14 was cloned;

S3: The promoter fragment of OsHEN1 obtained from S1 and the genomic DNA fragment of OsSPL14 obtained from S2 were connected to the plasmid vector linear fragment of pCAMBIA1305.1 double-digested by EcoRI and HindIII by homologous recombination, and transformed into E. PCR identification followed by sequencing identification to obtain a recombinant vector containing the genomic DNA fragment of OsSPL14 linked to the pOsHEN1::OsSPL14 expression cassette downstream of the OsHEN1 promoter fragment.

The homologous recombination system is as follows:

The recombination system was configured according to the above system, mixed with a pipette, placed at 37°C for 30 minutes, and then placed on ice for 5 minutes to carry out the next step of Escherichia coli transformation.

Transform E. coli

1. Take out a tube (100 μl) of competent bacteria from the -70°C ultra-low temperature freezer, and melt the competent bacteria in an ice bath for 5-10 minutes.

2. Add 5 μl of the ligated recombinant plasmid, shake gently and place on ice for 20 min.

3. Shake gently, insert it into a 42°C water bath for 90 seconds to heat shock, then quickly put it back on ice and let it stand for 2 minutes.

4. Add 500 μl of anti-LB medium to the above tubes in the ultra-clean workbench and mix gently, then fix it on the spring rack of the shaker and shake at 37°C for 45 minutes.

5. Take the above transformation mixture on the ultra-clean workbench, pour it into a solid LB plate petri dish containing kanamycin antibiotics, coat it with a glass coating rod burned by an alcohol lamp, and blow dry the surface of the medium in the ultra-clean workbench .

6. Invert the medium and put it into a 37°C constant temperature incubator overnight.

7. Pick a single clone colony for colony PCR identification, sequence and then identify the positive plasmid pOsHEN1::OsSPL14 vector.

Example 2 The recombinant expression vector containing the pOsHen1::OsSPL14 gene expression cassette was transformed into rice to obtain a transgenic line HS

The rice used for transformation was: wild-type rice Nipponbare (NIP).

A plurality of stable transgenic lines were obtained by the transformation method mediated by Agrobacterium oryzae, which were used for the experimental research of the present invention. The specific transformation, infection and transgenic rice cultivation methods are as follows:

1. Peel the mature seeds of rice and select healthy and intact seeds.

2. Wash and soak with 70% ethanol for 2-3 minutes, wash two to three times, and then wash with 10% sodium hypochlorite solution for 30 minutes.

3. Wash with sterile water three to four times.

4. Dry the peeled seeds on sterile filter paper.

5. Put the dried seeds on the induction medium, and cultivate in the dark for 15-20 days, until the larger yellow callus grows.

6. Peel off the callus and transfer to subculture medium for two weeks in the dark.

7. Co-cultivation. Agrobacterium EHA105 containing positive plasmid vector and callus were cultured in liquid co-culture medium with gentle shaking for 30 minutes, and the callus after co-culture was transferred to sterile filter paper to dry.

8. The air-dried callus is transferred to the co-culture medium and cultivated for 2-3 days.

9. Wash the callus with sterile water containing the antibiotic Timentin and transfer it to a screening medium containing 50 mg/L hygromycin, and cultivate in the dark for 2-3 weeks. Transfer to 50mg/L sieve medium and culture in the dark until new callus grows.

10. Transfer new callus to differentiation medium, and transfer to rooting medium after rice seedlings grow.

11. Transplant into the field when the rooting medium grows to strong seedlings.

Four transgenic lines HS-2, HS-9, HS-8, Hs-4 were obtained (Fig. 2).

Example 3 Research on agronomic characters of transgenic lines without induction by PXO99A

In this study, wild-type rice Nipponbare (NIP) was used as a control to study the agronomic characters of the four transgenic lines HS-2, HS-9, HS-8 and Hs-4 obtained in Example 2 without being induced by PXO99A.

3.1 Plant background OsSPL14 expression

Research method: real-time quantitative PCR

3.2 Trizol method to extract total RNA

Four transgenic lines HS-2, HS-9, HS-8, Hs-4 and NIP control were not induced by PXO99A, and the leaf samples were taken as follows:

(1) Weigh 0.1 g of fresh leaves, quickly grind them in liquid nitrogen with a mortar, thoroughly grind them, and quickly transfer them to a 1.5 mL centrifuge tube (precooled with liquid nitrogen). Add 1 mL of Trizol to the centrifuge tube and place on ice for 10 min;

(2) Add 100uL of chloroform, shake vigorously for 30S, and place on ice for 5min;

(3) 4℃, 13000rpm, 10min;

(4) Transfer the supernatant to another new 1.5ml RNase-Free centrifuge tube, add an equal volume of isopropanol, gently invert and mix, and place at room temperature for 10 minutes;

(5) 4℃, 13000rpm, 15min;

(6) Discard the supernatant, add 500uL RNase-Free 75% ethanol, and wash twice;

(7) pour out ethanol, place about 10min at room temperature;

(8) Add 50uL RNase-Free water, keep it in a metal bath at 65°C for 15 minutes, and store it at -80°C for later use;

3.3 RNA reverse transcription [HiScript II Q RT SuperMix for qPCR (+gDNA wiper) kit]

1. Total RNA 1ng-500ng

2. 2 microliters of gDNA wiper

3. Add sterile water to a total volume of 8 µl

The above mixture was placed in a PCR machine at 42 degrees Celsius for 2 min, then taken out and placed on ice. Add 2 microliters of HiScript II QRT SuperMix reverse transcriptase, in the PCR machine at 50°C for 15min, take it out after 2min at 85°C, dilute 200 times, and set aside. 3.4 Fluorescence quantitative PCR

Reaction system preparation

Reactor setup:

1. Pre-denaturation: 95°C for 5 minutes.

2. Cyclic reaction: 95°C for 10 seconds, 60°C for 30 seconds, 40 cycles.

3. Dissolution curve: 95°C for 15 seconds, 60°C for 30 seconds, and 95°C for 15 seconds.

Other detailed steps and real-time PCR are conventionally known formulas or kits commonly used in the market. There is no special treatment and will not be described in detail.

Fluorescence quantitative PCR gene number and primer sequence

The results showed that compared with the wild type, the expression of OsSPL14 in the background of the transgenic lines was up-regulated by 3-15 times in the absence of PXO99A induction (Fig. 3).

3.2 Plant height and tillers

The plant height and tiller status of four transgenic lines HS-2, HS-9, HS-8, and Hs-4 were compared with those of wild-type Nipponbare (NIP).

The results showed that compared with the wild type, the transgenic lines increased the plant height, decreased the number of tillers, and increased the number of effective tillers. (Fig. 4) 3.3 Thickness of the third stalk

The stem diameter of the third node of the four transgenic lines, HS-2, HS-9, HS-8, and Hs-4, was compared with that of wild-type rice Nipponbare (NIP).

The results showed that compared with the wild type, the stem diameter of the third node of the transgenic line increased and the lodging resistance was enhanced (Fig. 5).

3.4 Main spike

The primary branches, secondary branches and spikelet numbers of main spikes were compared between the four transgenic lines HS-2, HS-9, HS-8, and Hs-4 with wild-type Nipponbare (NIP).

The results showed that compared with the wild type, the primary branches of the transgenic lines did not change significantly, the secondary branches increased significantly, and the number of spikelets in the main panicle increased significantly (Figures 6, 7).

3.7 The number of main spikes

The number of main panicles of the four transgenic lines HS-2, HS-9, HS-8, and Hs-4 was compared with that of wild-type rice Nipponbare (NIP).

The results showed that compared with the wild type, the yield of the main panicle of HS-9 and HS-4 was significantly improved, but the yield of HS-2 and HS-8 had no obvious change. (Figure 8)

In conclusion, statistics show that these excellent yield traits affecting rice have excellent performance in transgenic rice HS lines.

Example 4 Fluorescence quantitative PCR identification of OsSPL14 in HS transgenic lines is specifically up-regulated by PXO99A induction

In this study, wild-type rice Nipponbare (NIP) was used as a control, and the four transgenic lines HS-2, HS-9, HS-8, and Hs-4 obtained in Example 2 were respectively inoculated with bacterial blight PXO99A 3 days later. The expression of OsSPL14 was determined to determine whether OsSPL14 was up-regulated by the OsHEN1 promoter under the specific induction of PXO99A.

Four transgenic lines, HS-2, HS-9, HS-8, and Hs-4, were injected and inoculated with bacterial blight PXO99A (OD value 0.5) on different leaves at the peak tillering stage, and were divided into different time points (0 h). , 72h) Sampling the inoculated leaves, the samples of each period were mixed with three leaves, and the total RNA was extracted respectively, which was used to identify the expression of OsSPL14.

4.1 PXO99A bacteria culture:

PSA medium

Peptone 10g/L

Sucrose 10g/L

Sodium glutamate 1g/L

Agar powder 12g/L

In a sterile ultra-clean bench, the bacterial liquid was evenly spread on the PSA medium plate, and after culturing in the dark at 28°C for 2-3 days, it was diluted with sterile water to the bacterial liquid OD600=0.5 for inoculation experiments.

4.2 Extraction of total RNA by Trizol method

Three transgenic lines and NIP control were inoculated with PXO99A in two inoculation periods, and leaf samples were taken for the following operations respectively to obtain the total RNA of the two inoculation periods:

(1) Weigh 0.1 g of fresh leaves, quickly grind them in liquid nitrogen with a mortar, thoroughly grind them, and quickly transfer them to a 1.5 mL centrifuge tube (precooled with liquid nitrogen). Add 1 mL of Trizol to the centrifuge tube and place on ice for 10 min;

(2) Add 100uL of chloroform, shake vigorously for 30S, and place on ice for 5min;

(3) 4℃, 13000rpm, 10min;

(4) Transfer the supernatant to another new 1.5ml RNase-Free centrifuge tube, add an equal volume of isopropanol, gently invert and mix, and place at room temperature for 10 minutes;

(5) 4℃, 13000rpm, 15min;

(6) Discard the supernatant, add 500uL RNase-Free 75% ethanol, and wash twice;

(7) pour out ethanol, place about 10min at room temperature;

(8) Add 50uL RNase-Free water, keep it in a metal bath at 65°C for 15 minutes, and store it at -80°C for later use;

4.3 RNA reverse transcription [HiScript II Q RT SuperMix for qPCR (+gDNA wiper) kit]

1. Total RNA 1ng-500ng

2. 2 microliters of gDNA wiper

3. Add sterile water to a total volume of 8 µl

The above mixture was placed in a PCR machine at 42 degrees Celsius for 2 min, then taken out and placed on ice. Add 2 microliters of HiScript II QRT SuperMix reverse transcriptase, in the PCR machine at 50°C for 15min, take it out after 2min at 85°C, dilute 200 times, and set aside.

4.4 Fluorescence quantitative PCR

Reaction system preparation

Reactor setup:

1. Pre-denaturation: 95°C for 5 minutes.

2. Cyclic reaction: 95°C for 10 seconds, 60°C for 30 seconds, 40 cycles.

3. Dissolution curve: 95°C for 15 seconds, 60°C for 30 seconds, and 95°C for 15 seconds.

Other detailed steps and real-time PCR are conventionally known formulas or kits commonly used in the market. There is no special treatment and will not be described in detail.

Fluorescence quantitative PCR gene number and primer sequence

The results showed that within 3 days of inoculation, PXO99A could induce the up-regulated expression of OsSPL14 in the HS transgenic lines, indicating that the OsHen1 promoter could significantly up-regulate the expression of OsSPL14 downstream under the specific induction of PXO99A by 2.75-3.26 times (Figure 9). .

Example 5 Study on disease resistance of transgenic lines in induced state

5.1 Lesion length

In this study, wild-type rice Nipponbare (NIP) was used as a control, and the four transgenic lines HS-2, HS-9, HS-8 and Hs-4 obtained in Example 2 were inoculated with Bacterial blight at the peak tillering stage of rice. The lesion lengths of PXO99A and J18 after 14 days were compared.

The results showed that the HS transgenic lines and their wild-type Nipponbare (NIP) were inoculated with PXO99A and J18 by leaf-cutting method at the peak tillering stage. 8. The lesion length of PXO99A in Hs-4 material was shorter than that of J18 by about 1.9 cm in the wild-type material, but the lesion length of PXO99A in the HS transgenic line was significantly shorter than that of J18 by about 3.0-3.8 cm (Fig. 10a, b); The study confirmed that the HS transgenic line has very strong specific resistance to rice bacterial blight PXO99A.

5.2 Resistance increase ratio

In this study, the four transgenic lines HS-2, HS-9, HS-8, and Hs-4 obtained in Example 2 were inoculated with bacterial blight PXO99A and J18 for 14 days after the rice tillering stage, respectively. Compared.

The ratio of increased resistance: (the length of the wild-type PXO99A lesions - the length of the lesions of the HS transgenic line PXO99A)/the length of the wild-type PXO99A lesions × 100% or (the length of the wild-type J18 lesions - the length of the lesions of the HS transgenic line J18) )/wild-type J18 lesion length × 100%.

The results showed that the resistance of the four transgenic lines HS-2, HS-9, HS-8, and Hs-4 to PXO99A was enhanced by 35%, 29%, 32% and 20%, respectively, while the four transgenic lines The resistance of HS-2, HS-9, HS-8, Hs-4 materials to J18 was enhanced by about 18%, 13%, 20% and 10%, respectively, which was significantly lower than that of PXO99A, the HS transgenic line The enhanced resistance to PXO99A was significantly higher than that of J18 by about 60-122%, and it was confirmed that the transgenic line could be specifically induced by Bacterial blight PXO99A to significantly increase the disease resistance (Fig. 10c).

sequence listing

<110> Nanjing Agricultural University

<120> pOsHEN1::OsSPL14 gene expression cassette and its construction method and application

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 49

<212> DNA

<213> Artificial Sequence

<400> 1

tatgaccatg attacgaatt cttatgtgca ctagaaacta tctgaggac 49

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 2

caaacgccca aaaaaaacaa 20

<210> 3

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 3

ttgttttttt tgggcgtttg ttccgtctct ttcctctctc ttct 44

<210> 4

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 4

tggtctttgt agtcaagctt cagagaccaa tccatcgtgt tg 42

<210> 5

<211> 1894

<212> DNA

<213> Rice (Oryza sativa)

<400> 5

gccactgaag taaacaagat gaaaaggaag catgcaggag gggcaggacc cttctggtgg 60

aaatggggga gtaaaaggct actgtacgaa gtgctatttg agggccgctg tctgaggaaa 120

accaaggata caggcaaaga aaaattgaat gatgaagcag agccctaaat gtgttgctgc 180

tgaagcctga ctgctgatct catcaacatc catagtgtag tgggctgact ggggttccct 240

gaatgtagct tgcatatacc gagccaaatt ttgactggag aattaagatg gtagttcgtg 300

tttttaggtg aaatttgtga acaaccttgt tgacatgtat tggatgttca gttgtattag 360

aaatagagtc aagtttacca tcctgtcgtc tgtcgaatga tcctgatgtt catttcaccg 420

cttcatttct gttgcctcgt tatcattttt tatgttaagc ctagcatatg gtattagcaa 480

accagcttga ttgatttttt gcatctctac tggaaattag gaactcaatt gctcatggag 540

ggaagaactc aaagatttgg gattctagct aactttacac agagtccaag ctgtttagga 600

tcctctgttg tgaactgaag agtattaaag agctaaacat gctcttgatc tgcaacagat 660

cagggcagtg ttgatggctc ctctcaccca ctgacccacc atctgtagtt tgttttgatc 720

ttaacctatt catcagcgct ataaagtaga actacggagt actccctcca ttccataata 780

taaggcataa tttatgttcc ataatataaa acatgtatgt atataggtaa ttaactgtga 840

tcccttctcc attaaattat tattttttta aatcctctac ttttatgtta tctaatttta 900

ttggatgcat gcattgtatt tattagaatg atcgaaacta caaaataata ataattattt 960

tcttgatctt ttggttaggg tggttatgcc ttatattttg gaatggaggg agtatttcat 1020

tattaacttc tatgtgcaac taggcaatag catctcagtc catggttttg cttaaactca 1080

cgttagttca gtttcgttaa tgtaaaatcc aatcaagcaa gagtatatgc atactgcatt 1140

cgcaaaaacc acttgaaaaa aaaaactgtg aaatatagag tagtgtttaa cattttactc 1200

ccttcggcca tttgacacaa tagttttaga ccatatagtt tgaccgttgc cttattcaaa 1260

agatttatgt aaatatcact ttttttaacct attttgttat taaaattagt ttagtatgac 1320

ttttatttttt acatatttgc actaaatttt taaataaaat gttctccctc ttttcttagt 1380

aattaacttt taaaaaatgt ttgaccattt atcttattta aaaattttat gcaaatgcat 1440

atattaaaaa acccagctag tttggtcaaa ctttgatgcg ttaaatattt atatacgagt 1500

gaaacgaccg tatttttcag aaaataatat aacaatcgat ttttacggga cataggcccg 1560

agattttctc aagggacact ttaccctcga aagagattct gcatttccgc taacgttaac 1620

aagaaggcgt attattggat gcagcgccat caaaaccttc tctccccctt cacacactcc 1680

ccctcgcttc ccttccctaa accccacttc acccgactcc tcgaatcctc ctcgcctcgc 1740

ttccgatttt tctggggggt ttagcgcctc ccccaattcg gcgaccaccc cctcccgatt 1800

ccgcctcagt tcctgcgtcg atcgattgcg aaaatcccct tcgttaccaa tcgcatttgt 1860

ttgtttgttt ttgtttgttt tttttgggcg tttg 1894

<210> 6

<211> 4770

<212> DNA

<213> Rice (Oryza sativa)

<400> 6

ttccgtctct ttcctctctc ttctctctcc ccctctcctg gaggagagag aggagaagag 60

gagggggggc cgcgccaaga gccacgcgcg ctacagtctc cttcccaccc gcgaccgcga 120

gcaatggaga tggccagtgg aggaggcgcc gccgccgccg ccggcggcgg agtaggcggc 180

agcggcggcg gtggtggtgg aggggacgag caccgccagc tgcacggtct caagttcggc 240

aagaagatct acttcgagga cgccgccgcg gcagcaggcg gcggcggcac tggcagtggc 300

agtggcagcg cgagcgccgc gccgccgtcc tcgtcttcca aggcggcggg tggtggacgc 360

ggcggagggg gcaagaacaa ggggaagggc gtggccgcgg cggcgccacc gccgccgccg 420

ccgccgccgc ggtgccaggt ggaggggtgc ggcgcggatc tgagcgggat caagaactac 480

tactgccgcc acaaggtgtg cttcatgcat tccaaggctc cccgcgtcgt cgtcgccggc 540

ctcgagcagc gcttctgcca gcagtgcagc aggtcactct ctcactcacc tcgccattgc 600

tgatgtcacc actgcttttg ctttgctttg cttgctctcc ctcctctttc acctatctct 660

cttgtttatt tgcttcttgt tcttgtttag tgctagtaca tgtgttgtta ttgttgtgcc 720

gttttgtctt ttgggttatt gtgttgttgt tactactcgt tttactatag gtttttaagg 780

tttatgagca cggccaccac attagatgca ctgtcaagtg gtgtgtgtgg gacctttcct 840

gctaaaacaa gctgatttca actctctgaa acttcctgca tttcatctat ttttatcttt 900

gattgtgttg ggagtactac actagtagtg ttaatatttt gactggtgct tatgagattt 960

ttaagttggt aggttgatga ggaaaatact cctttatatg gttgagtgat gtgacttgcc 1020

tgtctgcctg cctgcctgcc gctttgcata agattcctct gtgttagtaa gagccactgt 1080

ttatttgtac tggtgcttac tctacttagt taattagcca ttagctataa aattccgttg 1140

atgttgcaag cttagcaatg gccacggtaa gaatgggaga gagaagttgg ctaaagctgt 1200

tgctttgtag tttgtactat atatgtgtct ttgtgttgca agatatgcaa ctcctactat 1260

gctgtgactt gagctcaagg ttttcagtta tctatagatc cttactacta ctgagcatac 1320

taccacttct gtatggtagc atatggtagc atagtccaag ttccaacgcc tcgccagttg 1380

ttcataatct atactaccac ttctgtgcat ttgttacttt tatttaatag tttgtctcat 1440

tagctgacaa gcatatgcct gttttgatat ctgcccctct tgtaatagtc tatggatagc 1500

ttggactgtt tgatgcttta attttttact agcaacactt agggcccctt tgaaatggag 1560

gattagcaaa ggaattttgg aggattcatt ttcctaagga ttttttccta tagagccctt 1620

tgattcatag aaagaggata ggaaaacttc cgtaggattg cattcctatg atcaattcca 1680

taggaaaata agcaagaggt tagacctctt gtgaaacttt cctttgttga gtgtatcttg 1740

tggtataatc aaagggctct tctctccatt tcatgtgttt tcaattcctg taggattgga 1800

aaaacataca acttcaattc ctacgttttt cctattccta tgtttttcct atcctgcgtt 1860

tcaaaggggc ccttaaggat gaagggaagt aagagaaaca tactagagaa tatgtagtag 1920

tatttctaca ttccatattt gtagcactag cccacaaata tctttgcctt gtacttactt 1980

cataccagtt cccccctttt cagagcaaac caacaatttc tgttgcctta tatatctagt 2040

gtcttcgtac taatatatct gttccaaaat gtacctgtcc aaattcatag ctagaaatag 2100

ctttatttag gacggaagta ataactgttg ttagagactt ggttcagact tttggttatg 2160

ttgaggctac tatcatttcc tttacgggcc aaattactac aaatgagaat tcataaaaat 2220

gtcaagattt tatgattgtt gtagctttat ttaggacgga ggtagtaatt gttgttagag 2280

acttggttca gacttttggt tacgttgaag ctactatcat ttcctttatg gtcaaattac 2340

taacaatgag tattcataaa aatgtcaaga ttttataatt gagctgtgcc agtgctaagt 2400

gtgtcactat ctgatgccat aatgcatcat tataaaagcc agatggacca ttagctttta 2460

tgtgtaggac acctgccgtc caattagatg gataaccatc tagtgtttgt gtactgttat 2520

tttaagcccg acatctcaca actccatgaa tgattacagt cttcctttca catggtgtcc 2580

ttttgttgtg ttaggaatag cattttttat ttatgggtgt aattatgaaa ggcactagga 2640

gagttgctgc tttatcttga tgggatttgt agtaatacca tctttaggat gacaagaaat 2700

cttgttctga gttagcatgg gctgcctttt gacctgagct acggtttgct atgtttggct 2760

tgcatcatgc agatctatta ggataataag catataaaag ttgcttgcat tgtgcattgc 2820

ttgttttacc ttgattcatg taggagtaat ttgctcgcca tgcctcgttt tgctttctga 2880

gtcaacagcc aaatttagat gatgtacctt ctgttgcttc aaaaactcag tcactgcaca 2940

gcagcagtgg ataggattca gaatcaatct atccatgatt ctctgttcac ataatatgac 3000

aggttccacc tgctgcctga atttgaccaa ggaaaacgca gctgccgcag acgccttgca 3060

ggtcataatg agcgccggag gaggccgcaa acccctttgg catcacgcta cggtcgacta 3120

gctgcatctg ttggtggtat catcagaggc tcttgttttc tttgcatctt gtgtgtttgt 3180

tggtaactac tggttgcatt cgctgatgtg ttgtttgttg cgattcttga tccagaagag 3240

catcgcaggt tcagaagctt tacgttggat ttctcctacc caagggttcc aagcagcgta 3300

aggaatgcat ggccagcaat tcaaccaggc gatcggatct ccggtggtat ccagtggcac 3360

aggaacgtag ctcctcatgg tcactctagt gcagtggcgg gatatggtgc caacacatac 3420

agcggccaag gtagctcttc ttcagggcca ccggtgttcg ctggcccaaa tctccctcca 3480

ggtggatgtc tcgcaggggt cggtgccgcc accgactcga gctgtgctct ctctcttctg 3540

tcaacccagc catgggatac tactacccac agtgccgctg ccagccacaa ccaggctgca 3600

gccatgtcca ctaccaccag ctttgatggc aatcctgtgg caccctccgc catggcgggt 3660

agctacatgg caccaagccc ctggacaggt tctcggggcc atgagggtgg tggtcggagc 3720

gtggcgcacc agctaccaca tgaagtctca cttgatgagg tgcaccctgg tcctagccat 3780

catgcccact tctccggtga gcttgagctt gctctgcagg ggaacggtcc agccccagca 3840

ccacgcatcg atcctgggtc cggcagcacc ttcgaccaaa ccagcaacac gatggattgg 3900

tctctgtaga ggctgttcca gctgccatcg atctgtcgtc ccgcaaggcg agtcatggaa 3960

ctgaagaacc tcatgctgcc tgcccttatt ttgtgttcaa attttccttt ccagtatgga 4020

aaggaaattc taaggtgact ggcgattaat ctccctgtga tgaataataa tgcgcgccct 4080

tgaactcaat taattgctgt gccgcatcca tctatgtaac tctccatgaa tttttaagta 4140

tcagtgttaa tgctgtattg tcgaggactt ctgctcgata tgttatttct cttgttgt 4200

tcatcatgaa tctttttctg cttattattc tggtgccggg ttgtccttac cacagaagat 4260

tcagtttcgg ttggcgagag taaacacctt ccctggttgt gacaaaagct ccaacctttt 4320

cacttctcgg cctgtatttg atcttcccct tctgacgctg ttatactact tttaagcctg 4380

tatgtttcca gccttccagg tgaagggcca tactgaagag aaaacatgct ttcagggttt 4440

gatgcattgt gtactttaca agtgtactta agattttgta caatttatat atgtacctgc 4500

tctgctgctg agtattgtag gaaagaatca gttcgaaggg cgtgtgttca tgtaaagtga 4560

gaccacatgc acagcgtgga tttgcagcat gctctctgca ccagtggtgt tctgttgatg 4620

cctttgatgg gctggctgag gtgagaggag gatgatccat gttggcagct tcttcactct 4680

gaaaaataaa agagaagaaa tgttcagatt tgcagacaag tggagagcag tgatatattc 4740

tacaataaaa cattaccacc ttgcttttct 4770

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 7

cccagccatg ggatactact 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 8

tcaaagctgg tggtagtgga 20

<210> 9

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 9

aaccagctga ggcccaaga 19

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 10

acgattgatt taaccagtcc atga 24

Claims (6)

1. pOsHen1::OsSPL14 gene expression cassette, it is characterized in that, described pOsHEN1::OsSPL14 gene expression cassette comprises OsHEN1 promoter and OsSPL14 gene, and described OsSPL14 gene is connected with OsHEN1 promoter downstream, and described OsHEN1 promoter is based on rice. Nipponbare DNA was used as the template, and PCR amplification was carried out with OsHEN1 promoter-specific primers pOsHEN1-F: tatgaccatgattacgaattcTTATGTGCACTAGAAACTATCTGAGGAC (SEQ ID No. 1) and pOsHEN1-R: CAAACGCCCAAAAAAAACAA (SEQ ID No. 2), and cloned. 2. The recombinant expression vector containing the pOsHEN1::OsSPL14 gene expression cassette of claim 1. 3. The recombinant expression vector according to claim 2, wherein the recombinant expression vector is the EcoRI and HindIII sites that insert the pOsHen1::OsSPL14 gene expression cassette of claim 1 into the pCAMBIA1305.1 plasmid vector obtained in between. 4. the application of the pOsHEN1::OsSPL14 gene expression cassette described in claim 1 or any recombinant expression vector described in claim 2, 3 in improving rice crops, it is characterized in that, described improvement comprises: specifically improving rice new The resistance of the cultivar to bacterial blight PXO99A and/or increase the effective tiller number, and/or increase the stem diameter to enhance the lodging resistance. 5. the construction method of the recombinant expression vector described in claim 2, is characterized in that, described construction method comprises the steps: S1: Using rice Nipponbare DNA as a template, PCR amplification was performed with OsHEN1 promoter-specific primers pOsHEN1-F: tatgaccatgattacgaattcTTATGTGCACTAGAAACTATCTGAGGAC (SEQ ID No. 1) and pOsHEN1-R: CAAACGCCCAAAAAAAACAA (SEQ ID No. 2), and the promoter of OsHEN1 was cloned sub-amplification product; S2: Using rice Nipponbare DNA as a template, PCR amplification was performed with OsSPL14 gene-specific primers OsSPL14-F: ttgttttttttgggcgtttgTTCCGTCTCTTTCCTCTCTCTTCT (SEQ ID No. 3) and OsSPL14-R: tggtctttgtagtcaagcttCAGAGACCAATCCATCGTGTTG (SEQ ID No. 4), and the genomic DNA of OsSPL14 was cloned Amplification product; S3: The amplified fragments obtained from S1 and S2 were connected to the plasmid vector of pCAMBIA1305.1 double digested by EcoRI and HindIII by homologous recombination method to obtain a recombinant vector containing the pOsHEN1::OsSPL14 expression cassette. 6. according to the construction method of claim 5, it is characterized in that, described S3 concrete operation steps are: the pCAMBIA1305 after the promoter fragment of the OsHEN1 obtained by S1, the OsSPL14 obtained by S2 and the pCAMBIA1305 after EcoRI and HindIII double digestion .1 The linear fragments were connected together, transformed by E. coli, and the single clone was identified by colony PCR and then sequenced to obtain a recombinant vector containing the pOsHEN1::OsSPL14 expression cassette.

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