CN104711238B - A kind of Rice Drought Resistence albumen OsWS1 and its encoding gene and purposes - Google Patents

Abstract

The invention discloses a rice drought-resistant protein OsWS1, its coding gene and application. The present invention finds for the first time that the OsWS1 gene (its nucleotide sequence is shown in SEQ ID NO.1) is a member of the rice ultra-long-chain fatty acid synthase gene family. content, increase the thickness of the waxy layer on the leaf surface, thereby increasing the resistance of the plant to drought. Therefore, the OsWS1 gene can be used to control the tolerance of plants to drought and to cultivate plants such as rice drought-tolerant varieties.

Description

A kind of rice drought resistance protein OsWS1 and its coding gene and application

technical field

The invention belongs to the field of plant genetic engineering, and in particular relates to a rice drought-resistant protein OsWS1, its coding gene and its application.

Background technique

Rice (Oryza sativa L) is one of the most important food crops in the world and in China, and is the staple food of more than half of the world's population. At the same time, rice is also the crop with the largest water consumption. China's water consumption for rice production accounts for more than 65% of the total agricultural water use[ Mayra Rodriguez, Eduardo Canales, Carlos J. Borroto, eta1. Identification of genes induced upon water deficit stress in a drought-tolerant rice cultivar. Journal of Plant Physiology, 2006, 163: 577-584. Sasaki T, Burr B. International rice Genome sequencing projoct: theefort to completely sequence the rice genome. Cur Opin Plant Biol, 2000, 3(2): 138-141.]. As the trend of global warming intensifies, China, as a country with relatively scarce water resources, has become increasingly serious about water shortages. According to statistics, the reduction in rice production caused by drought can exceed the sum of the reductions caused by other factors, seriously affecting the yield and quality of rice [Hu Biaolin, Li Mingdi, Wan Yong, et al. Research progress on identification methods and indicators of early rice resistance in my country. Jiangxi Agricultural Journal, 2005, 17(2):56-60]. A large number of facts also reveal that the aridification in my country is intensifying, and the warming is significant. China's annual grain production reduction due to water shortage reaches more than 5 billion kilograms, the average annual drought-affected area reaches 26.37 million hm ² , and the economic loss caused by water shortage reaches 120 billion yuan. Studying the drought-resistant mechanism of rice and screening out drought-resistant rice varieties are not only of great significance to ensure rice production, but also have important guiding significance for improving water resource utilization.

Drought stress occurs in many parts of the world, which damages growth and severely restricts crop yield. Drought affects crop water uptake, mineral nutrient transport and metabolism, photosynthesis and distribution of photosynthetic products. All plants in nature are affected by drought, from commercial crops to field crops, aquatic crops to dry crops, and from lower organisms to higher organisms are affected by drought in varying degrees. Drought can start from seed germination and affect the life of plants.

Rice is an important field crop, its physiological ecology, flowering and fruiting, and grain quality are all damaged by drought stress. Drought affects almost all life activities of plants. However, plants have evolved a series of drought-resistant mechanisms to ensure that under water stress conditions, they can improve their drought-resistant ability through various channels. For example: reduce water loss by adjusting the size of stomata, produce osmotic adjustment substances to increase root water absorption, maintain plant metabolism by balancing peroxides in cells, maintain selective permeability of cell membranes, increase anti-stress hormones by reducing cell growth hormones increase its drought resistance. In addition, plants can also produce stress-resistance proteins, which increase the drought resistance of plants. Plants generally respond to drought stress through three forms of interaction: 1: cause changes in the expression of some genes; 2, produce or reduce the expression of some proteins to directly respond to drought stress to reduce damage to plants; 3, through physiological and biochemical Variations to enhance plant tolerance to drought.

At present, the cloning and transgenesis of rice drought-resistance-related genes have achieved initial results. The earliest transgenic plants related to rice drought resistance were developed by Xu et al. ,1996,l10(1):249-257.) Obtained, they introduced the HVAI gene into rice and obtained a large number of transgenic plants. Under drought stress, these transgenic lines can maintain high leaf relative water content, with little growth reduction, and HVAI protein can protect the cell membrane from damage, so that the growth rate is significantly better than that of the wild-type control, and the drought resistance is enhanced.

DREB can specifically combine with DRE cis-acting elements, and when plants are subjected to drought stress, it can activate the expression of stress-induced genes, so that plants can tolerate the effects of drought stress. Current studies have confirmed that OsDREB1A, OsDREB1B, OsDREB1C, OsDREB1D and OsDREB2A in the DREB transcription factor family can regulate the expression of genes related to plant growth and photosynthetic ability under drought stress, thereby improving the drought tolerance of plants (Mao Donghai, Chen Caiyan, Colinearity and Similar. Expression Pattern of Rice DREB1s Reveal Their Functional Conservation in the Cold-Responsive Pathway. PLoS ONE, 2012, 7(10):e47275). sNAcl is a transcription factor belonging to the NAM, ATAF and CUC (AC) family. Overexpression of sNAcl in rice can significantly increase the seed setting rate and yield of plants under drought stress (Honghong Hu, Mingqiu Dai, Jialing Yao, Benze Xiao , Xianghua Li, Qifa Zhang, Lizhong Xiong. Overexpressing aNAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. P NAS, 2006, 103(35): 12987-12992.)

During drought stress, regulatory genes encoding transcription factors such as DREB, MYB/MYC, and bZIP proteins bind to cis-acting elements in the promoter regions of corresponding drought-responsive target genes to activate gene expression, thereby regulating the response of plants to drought stress. adapt. Functionally related genes include LEA protein (late embryonic development abundant protein) gene, channel protein gene, osmotic regulation substance (such as proline, trehalose, betaine, polyamine, etc.) catalytic enzyme, etc.

Contents of the invention

The first object of the present invention is to provide an O-acyltransferase OsWS1 and its coding gene-drought resistance gene OsWS1 which can be used to improve plant drought resistance.

The patent of this invention takes a new fatty acid synthase gene OsWS1 of rice as the research object, constructs the OsWS1 gene overexpression vector OsWS1-OE, adopts the genetic transformation method mediated by Agrobacterium EHA105, and introduces the overexpression vector into the normal japonica rice variety Zhonghua 11, Finally, 42 tissue-cultured seedlings overexpressing the OsWS1 gene were obtained, and 16 tissue-cultured seedlings differentiated from normal japonica rice variety Zhonghua 11 as a control were planted in the experimental field. Compared with the control Zhonghua 11, the obtained T2 and T3 generation OsWS1 gene transgenic plants had an increase in the content of ultra-long-chain fatty acids in the leaves, an increase in the thickness of the wax on the surface of the outer skin, more and thicker verrucous protrusions, and chlorophyll fluidity and water loss rate. The drought resistance of plants increased significantly, indicating that the OsWS1 gene improved the drought resistance function of plants by affecting the content and structure of wax. The OsWS1 gene interference expression vector pTCK303-OsWS1-RNAi was constructed by RNAi technology, and the interference vector pTCK303-OsWS1-RNAi was introduced into the normal japonica rice variety Zhonghua 11 by using the genetic transformation method mediated by Agrobacterium EHA105, and finally the interference expression vector containing the OsWS1 gene was obtained Of the 24 tissue-cultured seedlings, 11 were identified as transgenic by PCR. The expression of OsWS1 gene was detected at the transcriptional level, and it was found that the expression of OsWS1 gene in the RNAi plants in T2 and T3 generation transgenic plants was significantly decreased. Moreover, the content of ultra-long-chain fatty acids in the leaves of the transgenic plants decreases, the wax on the surface of the outer skin becomes thinner, the verrucous protrusions become smaller and less, the fluidity of chlorophyll and the rate of water loss increase, and the resistance of the plants to drought is significantly reduced.

The O-acyltransferase OsWS1 of the present invention is characterized in that its amino acid sequence is shown in SEQ ID NO.2.

The second object of the present invention is to provide a drought resistance gene OsWS1, which is characterized in that its nucleotide sequence is shown in SEQ ID NO.1.

An expression vector containing the above-mentioned drought resistance gene OsWS1.

An engineering bacterium containing the above-mentioned expression vector.

The engineering bacteria is preferably Escherichia coli or Agrobacterium.

The third object of the present invention is to provide the application of the above-mentioned drought resistance gene OsWS1 in controlling the tolerance of plants to drought.

Preferably, the above-mentioned drought-resistant gene OsWS1 is used in breeding drought-tolerant varieties of plants.

Preferably, said plants include monocotyledonous plants and dicotyledonous plants.

Further preferably, the plant is rice, wheat, corn, cucumber, tomato, poplar, lawn grass or alfalfa.

The fourth object of the present invention is to provide an interference fragment for the drought-resistant gene OsWS1, which is characterized in that its nucleotide sequence is as follows: 5'-TGTTCTACTACATCACGCTGCGGCCGCCGACGGGGGAGGCGACCGCGTTCTTCACGCTGCACGGGGCGCTCGCCGTGGCGGAGGGGTGGTGGGCGGCGCGCGAGGGGTGGCCGCGGCCGCCGCCGCCGTCCGTCGCGACCGCGGCCCG'TCGACG.

The fifth object of the present invention is to provide the application of the above-mentioned interference fragment in regulating the expression of the drought resistance gene OsWS1.

The present invention finds for the first time that the OsWS1 gene is a member of the rice ultra-long-chain fatty acid synthase gene family. Overexpression of the OsWS1 gene can increase the content of ultra-long-chain fatty acids in rice leaves, increase the thickness of the waxy layer on the surface of the leaves, and increase the growth rate of the plant. Resistance to drought. Therefore, the OsWS1 gene can be used to control the tolerance of plants to drought and to cultivate plants such as rice drought-tolerant varieties.

Description of drawings

Figure 1 is a vector map of the constructed overexpression vector OsWS1-OE.

Figure 2 is the result of digesting with EcoRI after the cDNA of gene OsWS1 was ligated into pGEM T-easy vector, M is Marker, and 1 is pGEM T-easy containing gene OsWS1.

Figure 3 is the result of double digestion of the overexpression vector OsWS1-OE with HindIII and BamHI, M is Marker, and 1 is the overexpression vector OsWS1-OE after digestion.

Fig. 4 is a graph showing the PCR identification results of the overexpression vector OsWS1-OE Agrobacterium. M: marker; 1, 2, 3, 4 respectively represent a different clone.

Figure 5 is a diagram of the pTCK303 vector used in the interference vector experiment, a: a schematic diagram of the PTCK303 mother vector; b: a schematic diagram of the insertion of the OsWS1RNAi interference fragment.

Figure 6 is a diagram of the results of SpeI and SacI digestion and identification after the reverse fragment of the interference vector is inserted, M is a marker, and 1 is a sample.

Fig. 7 is a graph showing the identification results of the final vector of the interference vector by digestion with SpeI and SacI (1) and BamHI and KpnI (2).

Fig. 8 is the PCR identification result of interference vector Agrobacterium. M: marker; 1, 2, 3, 4 represent four different monoclonals respectively.

Figure 9 is a schematic diagram of the relative quantitative expression of OsWS1 in T2 transgenic plants, WT: control Zhonghua 11; OsWS1-ox: OsWS1 gene overexpression plants; OsWS1-RNAi: OsWS1 interference expression plants, 4-1, 6-4, 8- 4, 11-7, 14-4, 1-1, 4-14, 7-7, 8-1, 11-2 all represent different transgenic lines. From this result, it can be seen that the expression of OsWS1 gene was significantly increased in transgenic plants overexpressing OsWS1 gene, while the expression of OsWS1 gene was significantly decreased in OsWS1 RNAi transgenic plants.

Figure 10 is a co-localization result map of OsWS1: EGFP fusion protein, EGFP: control; ER-rk: ER marker protein, EGFP: EGFP fluorescence; OsWS1: EGFP: OsWS1 fusion EGFP carrier; merged: fusion of the two; from this result we can It can be seen that the expression of OsWS1 gene is localized in the endoplasmic reticulum, and the wax synthase it synthesizes is synthesized in the endoplasmic reticulum.

Fig. 11 is the relative quantitative expression results of OsWS1 gene during ABA and drought treatment. It can be seen from the results that the expression of OsWS1 gene was induced by ABA and drought, and the relative expression of OsWS1 gene gradually returned to normal level after drought-induced rehydration. A: Relative expression of OsWS1 gene after treatment with different concentrations of ABA for different time; B: Relative expression of OsWS1 gene during drought and rewatering.

Figure 12 is a comparison of the total content of wax components, fatty acid and long-chain fatty acid content in the leaves of different transgenic plants and control plants. A: Comparison of total wax content. The total wax content of OsWS1 overexpression plants was slightly increased compared with the control, while the total wax content of the interference expression plants was slightly decreased compared with the control. B: Comparison of long-chain fatty acids. The long chains from C20 to C34 in the leaves were measured by GC-MS method, and it was found that the content of long chain fatty acids in the overexpression OsWS1 transgenic plants was significantly higher than that of the control, while the long chain fatty acid content of the interference expression OsWS1 transgenic plants was significantly lower than that of the control . C: Comparison results of fatty acid content in plant leaves. It can be seen from the results that the contents of C16 and C18 fatty acids were significantly decreased in OsWS1 overexpression plants and significantly increased in RNAi transgenic plants. The content of long-chain fatty acids of C20-C34 was the opposite, and the content was significantly increased in transgenic plants overexpressing OsWS1, while the content was significantly decreased in RNAi plants. WT: control Zhonghua 11; OsWS1-ox: OsWS1 gene overexpression plant; OsWS1-RNAi: OsWS1 interference expression plant.

Figure 13 is the GC-MS measurement results of normal alkanes and aldehydes in different plants. A: GC-MS results of normal alkanes. As a result of the test, it was found that the content of normal alkanes from C23 to C32 did not change in the transgenic plants. B: GC-MS results of aldehydes. The test found that the content of extracted C26 to C34 aldehydes had no significant difference between the transgenic plants and the control. The results showed that the OsWS1 gene was only involved in the elongation of long-chain fatty acids. WT: control Zhonghua 11; OsWS1-ox: OsWS1 gene overexpression plant; OsWS1-RNAi: OsWS1 interference expression plant.

Figure 14 is the scanning electron microscope observation results of the waxy structure on the surface of plant leaves and stems. WT: control Zhonghua 11; OsWS1-ox: OsWS1 gene overexpression plant; OsWS1-RNAi: OsWS1 interference expression plant; Leaf: leaf; Stem: stem. It can be seen from the results. The verrucous granular waxy protrusions on the surface of leaves and stems of OsWS1 overexpressed transgenic plants were significantly larger and denser than those of controls (indicated by white arrows), while the verrucous granular waxy protrusions on the surface of leaves and stems of RNAi plants were significantly less and smaller than those of controls.

Fig. 15 is the transmission electron microscope observation results of the epidermis, mesophyll cells and pollen outer wall surface of the plant leaves. Adaxial surface: upper epidermis; Mesophyll cell: mesophyll cell; Pollen: pollen, CW: cell wall, WT: control flower 11; OsWS1-ox: OsWS1 gene overexpression plant; OsWS1-RNAi: OsWS1 interference expression plant. From the research results, we can see that the waxy layer of the outer epidermis on leaves of overexpressed OsWS1 transgenic plants is thicker and denser than that of the control (shown by black triangles), the thickness of mesophyll cells and pollen cell walls is relatively thin, and there are also Lots of underutilized grease on the walls (white star positions). The thickness of the cell wall in the RNAi transgenic plants becomes thinner, the texture is crisp, and there are many discontinuous small faults on the outer and inner walls of the pollen (shown by the black triangles with missing corners).

Figure 16 is a comparison of the leaf green extraction rate and water loss rate between OsWS1 overexpression transgenic plants and the control. A: Chlorophyll extraction rate. It can be seen from the results that the extraction rate of chlorophyll in the leaves of OsWS1 overexpressed transgenic plants was significantly slower than that of the control, but the opposite was true in RNAi plants. It was proved that the expression of OsWS1 gene affected the fluidity of chlorophyll in cells by affecting the distribution of wax on the surface of plant cell walls. B: Comparison of water loss rate. It can be seen from the results that the water loss rate of the leaves of overexpressed OsWS1 transgenic plants was significantly slower than that of the control, while the RNAi plants were on the contrary. It was proved that the expression of OsWS1 gene affected the water loss rate by affecting the wax distribution on the cell wall surface of the plant.

Figure 17 is a comparison experiment of drought tolerance between transgenic plants and controls. After the seeds germinated, several seedlings with the same growth were taken and grown normally in the culture pot. After 4 weeks (A) stop watering for 2 weeks (B) and then re-water, and after 3 days of re-watering (C) count the survival rate of the plants (D) . Three controls were done each time, and a total of 2 repetitions were carried out. From the statistical results, it can be seen that the overexpression OsWS1 plants show higher tolerance to drought than the control, while the drought tolerance of the RNAi plants is lower than that of the control, WT: control Zhonghua 11; OsWS1-ox: OsWS1 gene Overexpression plants; OsWS1-RNAi: OsWS1 interference expression plants.

Detailed ways

The following examples are to further illustrate the present invention, rather than limit the present invention.

Concrete experimental methods are not indicated in the following examples, and can be carried out according to conventional methods. The conditions are as described in J. Sambrook et al. "Molecular Cloning Experiment Guide", F. Osper et al.

Example 1: Functional Verification and Application of OsWS1 Gene

1. Construction of genetic transformation vector

The vector used for the overexpression vector is OsWS1-OE constructed in our laboratory. OsWS1-OE is a commonly used plant genetic transformation vector pCAMBIA3301 in the world (Sun et al.Xa26, a gene conferring resistance to Xanthomonas oryzae pv.oryzae in rice, encoding a LRR receptor kinase-likeprotein.Plant Journal.2004,37:517- 527), an Agrobacterium-mediated genetic transformation vector carrying a maize Ubiquitin promoter with constitutive and overexpression characteristics (Figure 1). The pCAMBIA3301 vector was purchased from the Australian CAMBIA Laboratory (center for the Application of Molecular Biology to International Agriculture). After replacing the 35S promoter with the maize ubiquitin promoter, it was named pXU3301. The technology for replacing the promoter is common knowledge, and those skilled in the art can Implemented according to conventional knowledge.

The RNA of rice Zhonghua 11 was extracted, reverse-transcribed into cDNA, and the drought-resistant gene OsWS1 (such as The nucleotide sequence shown in SEQ ID NO.1, the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2), and then connected to the pGEMT-easy vector through TA cloning, ligation reaction: PCR product 5 μ l, vector 0.5 μl, 2U T4ligase, 1μl of 10×buffer, total 10μl volume, ligation at 16°C for 3h. Take 10 μl of the ligation product, transfer it to E. coli top10 by calcium chloride freeze-thaw method, add 800ml LB, revive for 1 hour, centrifuge at 6000rpm for 5 minutes to enrich the bacteria, apply it on the LB plate of ampicillin antibiotic, and leave it overnight at 37°C. Pick a single clone, expand the culture to extract the plasmid, and identify it by enzyme digestion. The electrophoresis diagram of the enzyme digestion is shown in Figure 2. Sequencing was then performed, and it was found through sequencing that the drought-resistant gene OsWS1 (nucleotide sequence shown in SEQ ID NO.1) was inserted into the pGEMT-easy vector. After the sequencing was correct, the constructed vector was digested with HindIII and BglII and ligated to the large fragment of pXu3301 that had been digested (with HindIII and BamHI), and the ligated product was transformed into Agrobacterium competent EHA105. Plasmid was extracted, digested (enzyme digestion electrophoresis as shown in Figure 3) and PCR verification (PCR product electrophoresis as shown in Figure 4), and then sequenced, it was found through sequencing that the drought-resistant gene OsWS1 was inserted into the vector pXu3301, which was obtained by The constructed vector was thus obtained, named OsWS1-OE, 700 μl of Agrobacterium liquid containing the constructed vector was mixed with 300 μl of 50% glycerol, and stored at -70°C.

Interference vector The vector used is pTCK303-R1R2 constructed in our laboratory. pTCK303-R1R2 was rebuilt on the basis of pTCK303 (ZHEN WANG et al. A Practical Vector for Efficient Knock down of Gene Expression in Rice. Plant Molecular Biology Reporter. 2004,22:409-417), a commonly used plant genetic transformation vector in the world. Agrobacterium-mediated genetic transformation vector carrying Ubi promoter with constitutive and overexpression characteristics (Fig. 5). The pTCK303 vector was donated by the Research Center for Molecular and Developmental Biology (Research Center for Molecular and Developmental Biology) of the Institute of Botany, Chinese Academy of Sciences. The pTCK303 vector can also be purchased from a reagent company, which is a conventional vector in the prior art. Select the interval of the cDNA interference fragment of the drought resistance gene OsWS1 (nucleotide sequence shown in SEQ ID NO.1), use 481900-RNAi/F (GGGGTACCACTAGTCACTACATCACGCTGCGGC, KpnI and SpeI) and 481900-RNAi/R (GGATCCGAGCTCACCCCGTGGACATGACGAG, BamHI and SacI) primer amplification (PCR reaction system: cDNA 1μl, 10mM dNTPs 2μl; 481900-RNAi/F 2.0μl; 481900-RNAi/R 2.0μl; 10x E-Taq buffer 5.0μl; E-Taq 5U; total 50μl volume ；PCR扩增程序：94℃3min，94℃30'，56℃30'，72℃90'，35cycles，72℃10min)，将获得的cDNA片段(OsWS1 RNAi cDNA)( GGGGTACCACTAGTC TGATGTTCTACTACATCACGCTGCGGCCGCCGACGGGGGAGGCGACCGCGTTCTTCACGCTGCACGGGGCGCTCGCCGTGGCGGAGGGGTGGTGGGCGGCGCGCGAGGGGTGGCCGCGGCCGCCGCGCCCCGTCGCGACCGCGCTGACGCTGGCGCTCGTCATGTCCACGGGGT TCTGGCTCTTCT )连接pGEM T -easy vector, ligation reaction: PCR product 5 μl, vector 0.5 μl, 2U T4ligase, 10x buffer 1 μl, total volume 10 μl, ligation at 16°C for 3 hours. Take 10 μl of the ligation product, transfer it to E. coli top 10 by calcium chloride freeze-thaw method, add 800ml LB, revive for 1 hour, centrifuge the enriched bacteria at 6000rpm for 5 minutes, apply it on the LB plate of ampicillin antibiotic, and overnight at 37°C. Single clones were picked, expanded and cultured to extract plasmids, sequenced and identified. After the sequencing is correct, first digest with SpeI and SacI, connect the first cDNA fragment cut with SpeI and SacI to the large fragment of pTCK303 vector, transform Escherichia coli, extract the plasmid, and identify it by enzyme digestion (Enzyme digestion electrophoresis is shown in Figure 6). Select the large vector obtained by double digestion with BamHI and KpnI of the correct plasmid and the fragment of OsWS1RNAi cDNA (simultaneously digested with BamHI and KpnI) inserted into pGEM T-easy, ligate, transform into Escherichia coli, extract the plasmid and identify it by digestion. The correct plasmid was transformed into Agrobacterium competent EHA105, the plasmid was extracted, digested (the electropherogram of the enzyme digestion is shown in Figure 7) and PCR verification (the electrophoresis of the PCR product was shown in Figure 8), and the constructed interference vector was obtained. Named: OsWS1-RNAi. Take 700 μl of the Agrobacterium liquid containing the constructed vector, add 300 μl of 50% glycerol, mix well, and store at -70°C.

2. Genetic Transformation

The genetic transformation method mediated by Agrobacterium EHA105 (Hiei et al., Efficient transformation ofrice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant Journal, 1994,6: 271-282) will super The expression vector OsWS1-OE and the interference vector OsWS1-RNAi were introduced into the normal Zhonghua 11 rice variety respectively. The steps of Agrobacterium-mediated genetic transformation are as follows:

1. Callus induction: Ripe rice seeds are dehulled, then treated with 70% ethanol for 1 min, and sterilized with 5% sodium hypochlorite solution for 50 min; sterilized water washes the seeds 4-5 times; put the seeds on the induction medium; Cultivate in the dark for 5 weeks at a temperature of 25-27°C.

2. Callus subculture: select bright yellow, compact and relatively dry embryogenic calli, put them on the subculture medium and culture them in the dark for 2 weeks at a temperature of 25-27°C.

3. Pre-cultivation: select compact and relatively dry embryogenic calli, put them on the pre-medium and culture them in the dark for 4 days at a temperature of 25-27°C.

4. Agrobacterium culture: Pre-cultivate Agrobacterium EHA1052d containing the constructed vector on YEB medium with kanamycin and streptomycin at 28°C; transfer the Agrobacterium to the suspension medium and shake at 28°C Culture on the bed for 2-4h.

5. Agrobacterium infection: transfer the pre-cultured callus to a sterilized bottle; adjust the suspension of Agrobacterium to OD ₆₀₀ =0.8-1.0; soak the callus in the Agrobacterium suspension for 20 minutes; Injured until blotted dry on sterilized filter paper; then cultured on co-culture medium for 3 days at a temperature of 19-20°C.

6. Callus washing and selective culture: wash the callus with sterilized water until the Agrobacterium is invisible; soak in sterilized water containing 400 mg/L cephalosporin for 30 minutes; transfer the callus to sterilized filter paper and blot dry; Transfer the callus to selection medium for selection 2-3 times, each time for 2 weeks.

7. Differentiation and rooting: transfer the resistant callus to the dark place on the pre-differentiation medium and cultivate it for 5-7 days; transfer the callus of the pre-differentiation culture to the differentiation medium, and cultivate it under light (2000lx), at a temperature of 26°C, 5 -7 weeks.

8. Transplanting: After the callus differentiates into seedlings and takes root, wash off the residual medium on the roots, transfer the seedlings with a good root system to the greenhouse, and keep the water moist in the first few days.

9. Molecular biological detection of transgenic plants: After the transgenic plants grow up, cut the leaves to extract DNA, and design primers for PCR detection, and finally obtain 42 overexpressed tissue culture seedlings, harvest and plant , until homozygous plants were detected in the T2 generation, thus obtaining OsWS1 overexpression transgenic plants (OsWS1-OX). Similarly, primers were designed to perform PCR detection on OsWS1 interfering expression transgenic plants (OsWS1-RNAi), and finally 24 OsWS1 interfering expressing transgenic plants (OsWS1-RNAi) were obtained. The primers for detection of OsWS1 overexpression transgenic plants were bar-t/F: CACCATCGTCAACCACTAC; bar-t/R: GCTGCCAGAAACCCAC, and the PCR product fragment length was 431bp. The primers for detection of OsWS1 interference expression transgenic plants were hpt-t/F: GATGTTGGCGACCTCGTATT; hpt-t/R: TCGTTATGTTTATCGGCACTTT, and the PCR product fragment length was 517bp. A single plant was harvested and planted to obtain homozygous T2 and T3 generation plants, and the relative expression of OsWS1 gene in the transgenic plants was analyzed by quantitative PCR technique. The primers for quantitative PCR are: the internal reference gene is eEF-1a, the primer sequence is eEF-1aqRT/F: GCACGCTCTTCTTGCTTTC; eEF-1a qRT/R: AGGGAATCTTGTCAGGGTTG, the PCR product fragment length is 164bp; the primer sequence for OsWS1 is 40590-qRT/F: TCTGGGGACGGAGGTGGAA; 40590-qRT/R: AACATCAGCTCGTGCATGACG, PCR product fragment length 154bp. After analysis of quantitative PCR results of two generations of plants, it was found that the expression of OsWS1 in OsWS1 gene overexpression transgenic plants was stably and significantly increased, while in RNAi plants (OsWS1 interference expression transgenic plants (OsWS1-RNAi)) the expression was stably and significantly decreased (Figure 9 ).

3. Experimental verification of wax synthesis in OsWS1 transgenic plants

First, EGFP fusion protein expression combined with laser confocal observation experiment analysis found that OsWS1 and EGFP co-localized in the endoplasmic reticulum, proving that the long-chain fatty acid synthase synthesized by OsWS1 was synthesized in the endoplasmic reticulum (Figure 10). 1, 2, 4, 8, 12, and 24 hours after the treatment of 4-week-old rice seedlings with 0.5 μM and 5 μM ABA, the expression of OsWS1 gene was analyzed by quantitative PCR, and the analysis found that OsWS1 was induced by ABA (Figure 11- A). Take out the seedlings grown for 4 weeks in the rice hydroponic nutrient solution under normal conditions, absorb the water with absorbent paper, put them in the absorbent paper, and take samples at 0, 0.5, 1, 2, and 4 hours, and then place the seedlings in the hydroponic solution again Samples were taken at 0.5, 1, 2, 4, 8, and 12 hours, and the response of OsWS1 to drought was analyzed by quantitative PCR. It was found that OsWS1 was induced by drought, and its relative expression gradually returned to normal growth levels after rehydration (Figure 11- B).

According to the online analysis of OsWS1 homologous gene, it is a gene related to long-chain fatty acid synthase. In order to verify the function of the OsWS1 gene, various wax components in rice leaves at the four-leaf stage were analyzed by GC-MS. It was found that the total wax content in the OsWS1 gene overexpression transgenic plant material was slightly higher than that of the control, while the total wax content of the OsWS1 interference expression transgenic plant material was slightly lower than that of the control ( FIG. 12-A ). The total content of long-chain fatty acids (≥C20) in OsWS1 gene overexpression plants is now higher than that of the control, but in OsWS1 interfering expression transgenic plants is less than that of the control (Fig. 12-B). Comparing the content of long-chain fatty acids of C16-C34, it was found that the content of non-ultra-long-chain fatty acids C16 and C18 in the transgenic material overexpressing the OsWS1 gene was lower than that of the control, while the content of ultra-long-chain fatty acids greater than C18 was lower in OsWS1 The contents in the genetically modified materials were significantly higher than those in the control. The OsWS1 interference expression transgenic plants were the opposite, the content of C16 and C18 was higher than that of the control, and the content of ultra-long-chain fatty acids greater than C18 was lower than that of the control ( FIG. 12-C ). There was no difference in the expression of C23 to C32 n-alkanes and C26 to C34 aldehydes in leaves in the OsWS1 transgenic material. (Fig. 13-A, B) From the results of GC-MS, it can be seen that OsWS1 mainly affects the extension of ultra-long-chain fatty acids in plants, and does not participate in the extension of carbon chains of normal alkanes and aldehydes.

In order to further verify that the OsWS1 gene is involved in the elongation of ultra-long-chain fatty acids in plants and affects the composition and structure of the cell wall, we used transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to study the surface of leaves, stems and pollen. Waxiness was observed. It was found that the overexpression of the OsWS1 gene resulted in increased, larger, and thicker verrucous waxy protrusions covered on the surface of plant leaves and stems (Figure 14), and the cell walls of mesophyll cells were thickened. Lipid granules (Figure 15, indicated by white stars), pollen and mesophyll cells had thickened cell walls (Figure 15). However, RNAi interfered with the expression of OsWS1, and the verrucous waxy protrusions on the surface of leaves and stems of plants decreased and became smaller, and the cell walls were looser and thinner. Both the outer and inner walls of the pollen have discontinuous fine cracks (Fig. 14, 15). Through plant physiological experiments, the chlorophyll fluidity and water loss rate in the materials were compared, and it was found that the chlorophyll fluidity and water loss rate of the transgenic material overexpressing the OsWS1 gene were significantly slower than the control, while OsWS1 interfered with the chlorophyll flow in the transgenic plants. The sex and water loss rate were significantly faster than the control (Fig. 16A, B).

The above experiments all prove that the OsWS1 gene is a gene related to ultra-long-chain fatty acid synthase, which can affect the elongation of ultra-long-chain fatty acids in plants. Thus affecting the structure and composition of plant cell wall and cortex waxy components, and affecting the green fluidity and water loss rate of plants by affecting the waxy structure of the epidermis.

Further, we planted the plants in the experimental pots to conduct a drought tolerance experiment on the materials. In this experiment, a total of 2 biological repetition experiments were carried out, and each experiment had 3 repetitions. The specific results are shown in Figure 17. From the experimental results, we found that overexpression of OsWS1 gene can significantly improve the tolerance of plants to drought, while RNAi interference with the expression of OsWS1 gene can significantly reduce its tolerance to drought. The survival rate after drought rehydration was control ZH11: 54%; OsWS1-OE: 86%; OsWS1-RNAi: 29%.

Agrobacterium-mediated genetic transformation reagents and formulations

Reagents and Solutions Abbreviations

6-BA (6-BenzylaminoPurine, 6-benzyl adenine); KT (Kinetin, kinetin); NAA (Napthaleneacetic acid, naphthalene acetic acid); IAA (Indole-3-acetic acid, indole acetic acid); 2,4 -D(2,4-Dichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid); AS(Acetosringone, acetosyringone); CH(Casein Enzymatic Hydrolysate, hydrolyzed casein); HN(Hygromycin B, hygromycin) ; DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide); N6 macroelements; N6 trace elements; MS macroelements; MS trace elements

Solution formulations for tissue culture

1) N6 large amount of mother liquor [10 times concentrated solution (10×)]

Dissolve one by one, and then dilute to 1000ml at 20–25°C.

2) N6 trace element mother solution [100 times concentrated solution (100×)]

Dissolve at 20–25°C and make up to 1000ml.

3) Fe ₂ EDTA stock solution (100×)

Add 300ml of distilled water and 2.78g of iron sulfate (FeSO ₄ ·7H ₂ O) into a large Erlenmeyer flask

Add 300ml of distilled water to another large Erlenmeyer flask and heat it to 70°C, then add 3.73g of disodium ethylenediaminetetraacetic acid (Na ₂ EDTA·2H ₂ O)

After they are all dissolved, mix them together, keep them in a water bath at 70°C for 2 hours, adjust the volume to 1000ml, and store them at 4°C for later use.

4) Vitamin storage solution (100×)

Add water to make up to 1000ml, and store at 4°C for later use.

5) MS macroelement mother solution (10×)

Dissolve at 20-25°C and make up to 1000ml.

6) MS trace element mother solution (100×)

Dissolve at 20-25°C and make up to 1000ml.

7) 2,4-D stock solution (1mg/ml)

2,4-D 100mg

Dissolve 1ml of 1N potassium hydroxide for 5 minutes, then add 10ml of distilled water to dissolve completely, dilute to 100ml, and store at 20-25°C.

8) 6-BA stock solution (1mg/ml)

6-BA 100mg

Dissolve 1ml of 1N potassium hydroxide for 5 minutes, then add 10ml of distilled water to dissolve completely, dilute to 100ml, and store at 20-25°C.

9) NAA stock solution (1mg/ml)

NAA 100mg

Dissolve 1ml of 1N potassium hydroxide for 5 minutes, then add 10ml of distilled water to dissolve completely, dilute to 100ml, and store at 4°C for later use.

10) IAA stock solution (1mg/ml)

IAA 100mg

Dissolve 1ml of 1N potassium hydroxide for 5 minutes, then add 10ml of distilled water to dissolve completely, dilute to 100ml, and store at 4°C for later use.

11) Glucose storage solution (0.5mg/ml)

Glucose 125g

Dissolve in distilled water to a volume of 250ml, and store at 4°C after sterilization.

12) AS stock solution

AS 0.392g

DMSO 10ml

Aliquot into 1.5ml centrifuge tubes and store at 4°C for later use.

13) 1N potassium hydroxide stock solution

Potassium hydroxide 5.6g

Dissolve in distilled water to a volume of 100ml, and store at 20-25°C for later use.

14) KT stock solution (1mg/ml)

KT 100mg

Dissolve 1ml of 1N potassium hydroxide for 5 minutes, then add 10ml of distilled water to dissolve completely, dilute to 100ml, and store at 20-25°C.

Medium formula

1) Induction medium

Add distilled water to 900ml, adjust the pH value to 5.8 with 1N potassium hydroxide, boil (100°C) and set the volume to 1000ml, dispense into 50ml Erlenmeyer flasks (25ml/bottle), seal and sterilize.

2) Pre-medium

Add distilled water to 250ml, adjust the pH value to 5.8 with 1N potassium hydroxide, seal and sterilize.

Heat to dissolve the medium before use, add 5ml of glucose storage solution and 250μl of AS storage solution, and pour them into petri dishes (25ml/dish)

3) Suspension medium

Add distilled water to 100ml, adjust the pH value to 5.4, divide into two 100ml Erlenmeyer flasks, seal and sterilize.

Add 1ml glucose stock solution and 100ul AS stock solution before use.

4) Select medium

Add distilled water to 250ml, adjust the pH value to 6.0, seal and sterilize.

Dissolve the medium before use, add 250 μl of 50 mg/ml hygromycin and 500 mg/L cephalosporin, and pour them into petri dishes (25 ml/dish).

5) Pre-differentiation medium

Add distilled water to 250ml, adjust the pH value to 5.9 with 1N potassium hydroxide, seal and sterilize.

Dissolve the medium before use, add 250 μl of 50 mg/ml hygromycin and 500 mg/L cephalosporin, and pour them into petri dishes (25 ml/dish).

6) Differentiation medium

Add distilled water to 900ml, adjust the pH value to 6.0 with 1N potassium hydroxide, seal and sterilize.

Claims (2)

1. The application of the drought-resistant gene OsWS1 in affecting the elongation of ultra-long-chain fatty acids in rice. The nucleotide sequence of the drought-resistant gene OsWS1 is shown in SEQ ID NO.1. 2. The application of the interference fragment in affecting the elongation of ultra-long-chain fatty acids in rice. The nucleotide sequence of the interference fragment is as follows: 5'-TGATGTTCTACTACATCACGCTGCGGCCGCCGACGGGGGAGGCGACCGCGTTCTTCACGCTGCACGGGGCGCTCGCCGTGGCGGAGGGGTGGTGGGCGGCGCGCGAGGGGTGGCCGCGGCCGCCGCGCCGTCCGTCGCGACCGCGGCACGTCGTC.