CN110577938A - Application of ABA 8' -hydroxylase gene OsABA8ox2 in plant photomorphogenesis and root development - Google Patents

Abstract

The present invention relates to abscisic acid (ABA) 8' -hydroxylase genesOsABA8ox2The application in plant photomorphogenesis and root development belongs to plant gene engineering and genetic improvement. Rice ABA 8' -hydroxylase geneOsABA8ox2The amino acid sequence of the protein product is shown as SEQ ID No: 1 is shown. In the present invention, the gene is knocked out by gene editing technologyOsABA8ox2Then the photomorphogenesis under the condition of weak light or shading can be positively regulated and controlled, the shade-avoiding reaction is relieved, and the plant shade tolerance is improved; the root system structure under drought stress can be optimized to improve the drought tolerance of plants. This is achieved byThe character improvement has great application value for crop production and urban three-dimensional greening.

Description

ABA 8' -hydroxylase genesOsABA8ox2application in plant photomorphogenesis and root development

Technical Field

the invention relates to plant genetic engineering and genetic improvement, in particular to an ABA 8' -hydroxylase geneOsABA8ox2Application in plant photomorphogenesis and root development.

Background

Plants, as photoautotrophs, are very sensitive and smart to light environments. Photomorphogenesis and dark morphogenesis are two distinct sets of developmental programs adopted by plants to cope with ambient light (Trends in plant science, Xu et al 2015). In the southwest region of China, Sichuan and Guizhou provincesAll the year round under weak light (Chinese oil crop institute, Van Yuan Fang, etc., 2016). Continuous or frequent rainy and short-time weather in the areas such as Huang-Huai-Hai brings very adverse effect on the production of crops such as rice, corn and the like. Close planting or intercropping adopted in modern agricultural production can aggravate competition of crop groups for light. With the acceleration of urbanization process in China, a lot of land forms a shading environment because of being surrounded by buildings. In a word, weak illumination or shading conditions affect photomorphogenesis of non-shade-tolerant plants, shade-avoidance reactions (over-extended growth, slender stems, easy lodging and the like) often occur, and plant growth, stress resistance, final quality and yield are affected. Therefore, the method for improving the plant shade tolerance has great application value. Plant shade tolerance refers to the ability of a plant to live under low light conditions, is a series of changes of the plant to adapt to low light quantum density and maintain normal growth and development of the plant (tropical agricultural science, anfeng and linjifu, 2005; Anhui agricultural science, Hui Dynasty, etc., 2015), and is an important trait of the plant. Plants will adopt two countermeasures in the face of weak light and shade: shade avoidance response and shade tolerance response (molecular plant breeding, luobiao, etc., 2019). The elongation reaction is one of the main manifestations of the shade-avoidance reaction (Current Opinion in Plant Biology, Fraser et al, 2016). Compared with the plant with weak shade-tolerant ability, the plant with strong shade-tolerant ability has relatively unobvious elongation reaction. One of the mechanisms of PIF (phytochrome-interacting factor) protein to promote elongation reaction is that it mediates the elongation reactionYUCCAGene up-regulated expression (Genes)&Development, Li et al, 2012), increased auxin synthesis. Antagonistic factors for The shade-avoidance response, such as PAR1 and PAR2 proteins (The EMBO Journal, Roig-Villanova et al 2007) are also present. In addition to the use of shade-tolerant genes, a regulatory pathway that suppresses the shade-avoidance response may be another mechanism for generating a shade-tolerant response (Current Opinion in plant Biology, vandenbusche et al, 2005). The function of down regulating/inhibiting the shade-avoiding gene is one of the technical ideas for improving the shade tolerance of plants. Current research is mainly focused on the response of plants to light signals, such as the response of the phytochrome interaction factor PIF to the changing light environment through the phytochrome signaling pathway (phyB-PIF) (Plant physiology,Wuet al, 2019). The main mechanism of shade-tolerant plants adapting to low-light environments is the maximized absorption of light quanta for photosynthesis (the Hunan forestry science, Huangdi and Wu-Tieming, 2011). The crossing of light with the ABA signaling pathway has been reported in arabidopsis thaliana. BBX21, a B-box protein, regulates photomorphogenesis and also participates in the ABA signaling pathway (PLoS genetics, Xu et al 2014). HY5 isABI5（ABA INSENSITIVE 5) Direct activators of transcription. BBX21 interference by HY5 withABI5Binding of promoters, negative regulationABI5And (4) expressing. ABA decomposition is one of important ways for regulating the concentration of ABA in plants. ABA 8' -hydroxylase is a key enzyme in the ABA degradation pathway and consists ofOsABA8ox1, OsABA8ox2AndOsABA8ox3Code (Plant)&cell physiology, Saika et al, 2007). Our country's study of plant shade tolerance started in the 70's of the 20 th century, and started late (the science of Hunan forestry, Huangdi and Wu-Tieming, 2011). At present, the plant shade-tolerant molecular mechanism is still unclear, and particularly, the related research on participation of key genes in ABA decomposition pathway in photomorphogenetic establishment, shade-avoiding reaction and shade-tolerant regulation is lacked.

Extreme weather conditions occur more and more frequently as the world climate warms, with drought being one of them. Drought stress (water deficit) can severely limit agricultural production. Water availability is decisive for plant survival and adequate growth. Root elongation growth to reach deeper soil layers to absorb sufficient moisture and nutrients is critical to the survival and growth of most plants. Root architecture is therefore critical to plant survival. Plants with strong drought tolerance can adapt to water-deficient soil environments by properly optimizing root system structures. Therefore, important root structure forming genes are excavated and applied to enhance the drought tolerance of crops, and the gene has important value under the trend of more frequent extreme weather. When the illumination is insufficient, the plant becomes thin and weak, the leaves become long, narrow and thin, the photosynthetic products conveyed downwards are reduced, the root growth is influenced, and the root system becomes shallow (tropical agricultural science, anfeng and linzifu, 2005). And within a certain range, as the shading degree is increased, the ratio of roots in the total weight of the whole plant is reduced, and the number of leaves is increased. So that the photomorphogenesis under weak light is positively regulated and controlled, and the improvementThe plant shade tolerance is very beneficial to the growth and development of plant roots and the optimization of root system structures. ABA treatment significantly increased the elongation of the main root and root hair density after 24 hours, whereas FLU (fluridone, fluazinone, ABA synthesis inhibitor) treatment was significant inhibition (New Phytologist, Xu et al, 2013). Arabidopsis ABA synthetic mutantsaba3-1Is a severe reduction compared to wild type, indicating the effect of ABA on root tip response under moderate water stress (New Phytologist, Xu et al, 2013). Compared with the wild rice seedlings, the method has the advantages that,OsABA8ox3 RNA interference (RNAi) transgenic lines have greatly improved drought stress tolerance (PLoS One, Cai et al. 2015), but lack the research results of root development and root structure of RNAi strains. The master academic paper of Hakka research on producing Kouyol (Applicant) ((OsABA8ox2RNAi transgenic rice identification and ABA-related gene expression analysis, basic science editions of the "full-text database of Chinese Excellent Master thesis", A006-307, 20150115) points out the three-leaf stageOsABA8ox2RNAi transgenic rice treated with 20% PEG6000 had significantly higher survival than wild type, but in the method the following are written: "20% PEG6000 simulates drought stress: 20g PEG6000 dissolved in 1L culture solution. ". Whether 20% PEG6000 or 2% is problematic, and root development and root structure of RNAi transgenic rice have not been studied. The RNAi technology is used for reducing gene expression and functions, and the accuracy and the flexibility of the RNAi technology are far inferior to those of the current gene editing technology for fire and heat development. U.S. Pat. No. ' nucleic acids modified Dormancy ' (patent No.: US 8269082B 2; patent date: 9/18/2012) discloses polypeptides having ABA8 ' -hydroxylase activity and polynucleotides encoding these proteins affect seed germination rate and Dormancy.

In summary, at present, there is no solution for the problemOsABA8oxThe family genes are involved in the report of plant photomorphogenesis and root development.

Disclosure of Invention

To solve the above problems, the present invention provides an ABA 8' -hydroxylase geneOsABA8ox2application in plant photomorphogenesis and root development.

The technical scheme provided by the invention is as follows: by inactivating plantsIn vivo ABA 8' -hydroxylase genesOsABA8ox2The function of relieving the shade-avoiding reaction of plants, improving the shade tolerance and optimizing the root system structure under drought stress.

FromOsABA8ox2a specific editing target sequence (SEQ ID No: 6) of 19bp is selected from a coding sequence (SEQ ID No: 4), and a T4 DNA Ligase (NEB, Ipswich, MA, USA) is utilized to construct a knockout vector pHUN4c12-OsABA8ox2. Rice strains with target sequences inserted with 1 base and deleted with 2 bases are obtained through agrobacterium-mediated transformation. The mutated target sequence is shown in SEQ ID No: 7 and SEQ ID No: shown in fig. 8.OsABA8ox2A frame shift mutation is generated in a coding sequence (CDS) near the 5' end, and the amino acid sequence after mutation is shown as SEQ ID No: 2 and SEQ ID NO: 3, respectively. All homozygosityOsABA8ox2Compared with wild rice, the knockout strain shows that the plant height is reduced, the leaves are shortened, the shade tolerance is enhanced and the root system is optimized. The OsABA8ox2 protein has an amino acid sequence shown in SEQ ID No: 1. The plant is monocotyledon, but also can be dicotyledon. In the embodiment of the invention, the plant is monocotyledonous rice, in particular to japonica rice variety Kitaake.

Will be completeOsABA8ox2The coding sequence (SEQ ID No: 4) is integrated by homologous recombination into the engineered oneBamHI (NEB) andSacThe pCAMBIA1303 vector after double digestion of I (NEB) was subjected toUbi-1And (3) a promoter driver. Against the background of Kitaake obtained by Agrobacterium-mediated transformationOsABA8ox2Overexpression of transgenic rice lines. Compared with wild type, the leaf elongation and plant height of the over-expression strain are obviously increased, and the shade-avoiding reaction is enhanced; the root elongation is severely inhibited and the total root length is significantly shortened.

Will be provided withOsABA8oxConstruction of pCAMBIA 1303-containing gene by fusing family 3 promoters (SEQ ID No: 5, SEQ ID No: 9 and SEQ ID No: 10) with beta-Glucuronidase (GUS)PRO _OsABA8ox :GUSand the vector is used for obtaining a transgenic rice strain with Kitaake as background by utilizing agrobacterium-mediated transformation. Analyzed by GUS stainingOsABA8oxFamily 3 promoter Activity, in particularOsABA8ox2the promoter activity of (1).OsABA8ox2Strong expression in the root meristematic region during seedling stage. Has strong expression in glumous flowers and seeds in the filling stage.

The invention has the beneficial effects that:

1. Obtained by the Gene editing techniqueOsABA8ox2The rice plant line is knocked out, under a certain weak light condition, the shade-avoiding reaction is not obvious, the shade tolerance is obviously improved, and the rice plant photomorphogenesis is good.

2. Obtained by the Gene editing techniqueOsABA8ox2The rice strain is knocked out, and compared with wild rice, a root system structure which is more suitable for a water-deficient environment is developed under the soil drought stress condition within a certain period of time, so that the rice strain has higher survival capability.

3. OsABA8ox2Not only is a newly discovered shade-avoiding gene, but also is a new gene related to the formation of root system structure, thereforeOsABA8ox2The scientific and reasonable application in plant photomorphogenesis and root development has great value on the genetic improvement of gramineous crops such as rice and other plants and has wide application prospect.

4. OsABA8ox2Strong expression in seedling root meristematic region and strong expression in glumous flower and filling seed.OsABA8ox2The promoter of (a) has strong promoter activity in root meristems, glume flowers and seeds at the filling stage.

Drawings

FIG. 1OsABA8ox2Identification of knock-out (KO) rice lines.

FIG. 2OsABA8ox2Identification of over-expressed (OE) rice lines.

FIG. 3OsABA8ox2And (4) Knocking Out (KO) and over-expressing (OE) rice lines, and performing photomorphogenesis phenotype under weak illumination, and counting results of leaf length and plant height. Note significant differences (Student's) between KO (or OE) shoots and wild typettest，**P < 0.01）。

FIG. 4-Natural Low light conditionsOsABA8ox2Knock-out (KO) rice lines exhibit high shade tolerance, and over-expressed (OE) lines exhibit strong shade-avoidance response.

FIG. 5-ABA and IAA content in leaves. Asterisk indicates KSignificant differences between O (or OE) shoots and wild type (Student's)t test，* P < 0.05，** P < 0.01）。

FIG. 6-phenotypic observations of roots.

FIG. 7OsABA8ox2Knock-out (KO) and over-expressed (OE) rice lines were analyzed for drought tolerance. Note significant differences (Student's) between KO (or OE) shoots and wild typet test，** P < 0.01）。

FIG. 8OsABA8oxAnd (3) carrying out GUS staining analysis on the promoter and beta-Glucuronidase (GUS) fusion expression transgenic rice line.

FIG. 9OsABA8ox2And (3) performing GUS staining analysis on the promoter and GUS fusion expression transgenic rice line.

Sequence List description

SEQ ID No：1 — OsABA8ox2The amino acid sequence of the protein product,

SEQ ID No：2 — OsABA8ox2The amino acid sequence of the mutein product,

SEQ ID No：3 — OsABA8ox2The amino acid sequence of the mutein product,

SEQ ID No：4 — OsABA8ox2A coding DNA sequence which is capable of coding,

SEQ ID No：5 — OsABA8ox2A promoter sequence which is capable of being used as a promoter,

SEQ ID No: editing the target sequence by 6-19 bp,

SEQ ID No: 7-the target sequence of the mutation,

SEQ ID No: 8-the target sequence of the mutation,

SEQ ID No：9 — OsABA8ox1a promoter sequence which is capable of being used as a promoter,

SEQ ID No：10 — OsABA8ox3a promoter sequence.

Detailed Description

The present invention will be described in detail below by way of examples with reference to the accompanying drawings, but the present invention is not limited thereto and is only described by way of example.

Example 1

OsABA8ox2Construction of knock-out vectors and Gene editing plants（OsABA8ox2Knock-out rice line) obtained

A,OsABA8ox2Knock-out vector pHUN4c12-OsABA8ox2Construction of

A specific target sequence of 19bp was selected for the generation of sgRNA (single-guide RNA) using E-CRISP (http:// www.e-CRISP. org/E-CRISP/designrispr. html). The sequence (SEQ ID No: 6) is located inOsABA8ox2The CDS sequence (SEQ ID No: 4) of 1533bp in length is 92-110 bp (5'-GGAGGAGAGATGTTGGACA-3'). Synthesis of primer pairsOsABA8ox2-CRIS(Table 1), the phosphate group was added to the 5' end of the sequence using the following system:

OsABA8ox2-CRISF (10 mM) 1μl

OsABA8ox2-CRISR (10 mM) 1μl,

10x T4 DNA Ligase Buffer (with 10 mM ATP) 2μl,

T4 polynucleotide kinase (3’ phosphatase plus；NEB) 1μl,

H₂O 15μl.

40 min at 37 ℃, 20 min at 65 ℃ (enzyme heat inactivation), 5 min at 94 ℃ and 2 min at 50 ℃.

Plasmid pHUN4c12 (In Vitro Mutagenesis, Xu et al 2017)BsaI (NEB) is cut by the following system:

pHUN4c12 3μl (0.3μg/μl),

10x CutSmart Buffer 2μl,

BsaⅠ (NEB) 1μl,

H₂O 14μl.

3 tubes in total. 5 h at 37 ℃ and 20 min at 65 ℃ (enzyme heat inactivation). Adding 60 μ l of 3-tube enzyme digestion solution to 200 μ l, adding 2.5 times of anhydrous ethanol, mixing, and standing at-80 deg.C for 1 h. Centrifuging at 12000 rpm for 12 min, air drying, and adding 20 μ l H₂And dissolving the O.

Then adding phosphoric acid groupOsABA8ox2-CRISThe sequence was ligated to the above-described digested pHUN4c12 vector by T4 DNA Ligase (NEB).

II, gene editing plants: (OsABA8ox2Knock-out rice line) obtained

The constructed knock-out vector pHUN4c12-OsABA8ox2Transfer into Agrobacterium EHA105 (pSoup) competent cells (2) according to the protocol of the product (heat shock method)^ndLab, shanghai). The rice transformation work was entrusted to the division of the crop design by the unknown Xingwang system, laboratory (Beijing) Co. Briefly described as follows: will contain pHUN4c12-OsABA8ox2The agrobacterium EHA105 (pSoup) infects calluses of japonica rice varieties Kitaake, the calluses are transferred to a co-culture medium for 2 to 4 days in a dark way at 24 ℃, the cleaned calluses are transferred to a selection culture medium containing hygromycin for resistance screening, the selected resistance calluses are transferred to a pre-differentiation culture medium for 7 to 10 days, then are transferred to a differentiation culture medium for illumination culture, and are transferred to a rooting culture medium for about 3 weeks when seedlings grow to 2 to 4 cm. Well-grown plantlet (T)₀And) hardening seedlings for 3 days, and then transplanting the seedlings into soil. The seeds are received and then planted to obtain T₁And (4) generation.

III,OsABA8ox2Identification of knock-out Rice lines

First, T is identified₀whether the generation knockout rice line is integrated into the rice line containingOsABA8ox2-CRISThe T-DNA fragment of (1). With wild type and T₀Using the genome DNA of seedling-substituting leaf as templateU3bF/UbiR(Table 1) PCR (polymerase chain reaction) amplification was performed, and the plants with specific amplification bands were positive for the transgene (FIG. 1A). Specific primers designed on two sides of rice genome target sequenceOsABA8ox2-target(Table 1), PCR amplification (FIG. 1B) was performed, and T was detected by sequencing₀Surrogate target sequence information. 8 heterozygous mutants and 4 homozygous mutants were obtained, of which 2 homozygous mutants, KO1 and KO2, were shown in FIG. 1C, and the target sequences of the mutations were shown in SEQ ID Nos: 7 and SEQ ID No: shown in fig. 8. KO1 inserted 1G between 107 and 108bp of the CDS sequence of 1533bp in length. TG deletions at 105 and 106bp in CDS of KO 2.OsABA8ox2The CDS mutation site is close to the 5' end of the CDS mutation site, and a frame shift mutation occurs, and the amino acid sequence after mutation is shown as SEQ ID No: 2 and SEQ ID No: 3, OsABA8ox2 function was deleted. T is₁The same method was used for the detection.

TABLE 1 primers used in the examples

Example 2

OsABA8ox2Obtaining and identifying over-expressed rice lines

To build upOsABA8ox2Over-expression vector, completeOsABA8ox2The coding sequence (SEQ ID No: 4) adopts a primer pairOsABA8ox2-OE(Table 1) RT-PCR (reverse transcription polymerase chain reaction) amplification was performed. The amplified fragments verified by sequencing are integrated into the engineered fragments by homologous recombination methods andBamHI (NEB) andSacThe pCAMBIA1303 vector after double digestion of I (NEB) was placed inUbi-1The 3' end of the promoter is driven by the promoter. The GBclone Kit (GBI, Suzhou) was selected for homologous recombination. As in example 1, overexpression transgenic lines of japonica rice variety Kitaake as background were obtained using Agrobacterium-mediated transformation. Using genome DNA as template and primer pairOsABA8ox2-C(Table 1) amplification of exogenous DNA by PCROsABA8ox2CDS fragments were used to identify positive transgenic lines (FIG. 2A). The quantitative RT-PCR results in FIG. 2B show overexpression of transgenic lines OE1 and OE2OsABA8ox2The transcription level is far higher than that of Wild Type (WT), and the primer pair isOsABA8ox2-RT(Table 1), internal control isActin1(primers are shown in Table 1).

Example 3

OsABA8ox2Knocking out rice line photomorphogenesis phenotype, measuring leaf length and plant height and analyzing shade tolerance

plants were grown under weaker (2100 lux) lighting conditions. In comparison with the Wild Type (WT),OsABA8ox2The third leaf and sheath of the over-expressed (OE) strain were slender and weak (fig. 3A), in particular the length of the sheath was significantly higher than the wild type (fig. 3B), and the third leaf of the knock-out strains KO1 and KO2 was significantly shorter (fig. 3A, B). OE leaves are lighter in color than wild type, light green, while KO leaves are dark green.OsABA8ox2Participates in the development of leaves. 25-day-old WT seedlings were in the 3-leaf stage, while OE seedlings had extended in the 4 th leaf (FIG. 3A), and the over-expressed seedlings grew faster than wild-type. Due to the fact thatOsABA8ox2OE seedlingThe leaves were slender, the sheath was long and delicate, and the plants could not grow straight, curving to one side (fig. 3A). InferenceOsABA8ox2The over-expression inhibits the photomorphogenesis of the rice seedling stage and presents shade-avoiding reaction. The statistical result of plant height shows thatOsABA8ox2Both over-expressed lines OE1 and OE2 were significantly higher than wild type, whereasOsABA8ox2Both knock-out rice lines KO1 and KO2 were significantly shorter than wild-type (fig. 3B). Leaf elongation and plant height are important index traits for judging shade-avoiding response and shade tolerance of plants. Accordingly, the judgment is madeOsABA8ox2The knockout rice strain has stronger shade-tolerant capability than the wild type. In addition, a primer pair is usedCOP1(Table 1) semi-quantitative RT-PCR analysis was performed to find Arabidopsis thalianaCOP1Homologous genes in rice (accession number LOC _ Os02g 53140) are described inOsABA8ox2The transcriptional level decreased in knockout rice lines (FIG. 3C). In Arabidopsis thalianaCOP1Encodes an inhibitor of photomorphogenesis (Cell, von Arnim and Deng 1994; Trends in plant science, Lau and Deng 2012). The homologous gene is inOsABA8ox2The decrease in transcription level in the knockout rice line suggestsOsABA8ox2And (3) knocking out the forward regulation and control of the photomorphogenetic molecular network in the rice strain.

Under the condition of natural weak lightOsABA8ox2Knockout (KO) rice lines exhibited high shade tolerance and maintained straight growth (fig. 4). Whereas the over-expressed (OE) strain and wild type showed varying degrees of shade-avoidance response and could not maintain a straight growth (fig. 4). Here, the natural low light condition is: indoor, there is glass to introduce illumination to its east and north. 10:00, 13000 lux, 11:00, 2400lux in the morning; noon 12:00, 1800 lux; at 15:00, 700 lux, 17:00, 300 lux in the afternoon.OsABA8ox2To avoid shade gene. After OsABA8ox2 function inactivation, the plant is better constructed in a photomorphogenesis under a certain degree of weak light, and the plant shade tolerance is improved (figure 3A, figure 4).

Example 4

Determination of ABA and 3-indoleacetic acid (IAA) content in leaves

The determination of ABA and IAA content of leaves is finished by Hangzhou Jingjie biotechnology limited company, and an ultra-performance liquid chromatography/tandem quadrupole mass spectrometry system (ultra-performance liquid chromatography/tandem chromatography) is adopted for determining the length of the leavestandemquadruple mass spectrometer with an electrolytic interface, UPLC-ESI-qMS/MS). 5 leaf stage compared with wild typeOsABA8ox2KO1 functional leaves contained higher ABA levels, whereas OE2 leaves contained lower ABA levels and higher IAA levels (fig. 5).OsABA8ox2higher levels of IAA in leaves of over-expressed lines promote elongation reaction, which is one of the reasons why leaf elongation and plant height performance are prominent.

Example 5

Root phenotype observation and root parameter determination

ImageJ (https:// ImageJ. nih. gov/ij /) was used to measure the adventitious root diameter. The roots were weighed after 24 h exposure to room temperature. Whether under control conditions or drought treatment,OsABA8ox2OE2 was less than wild type in all root length, adventitious root diameter, and total root weight (fig. 6, table 2). The number of adventitious roots of KO1 significantly decreased after 2 weeks of stopped watering (table 2). The indefinite number of roots with a total root length of KO1 of 70% or greater under drought stress was significantly greater than wild type (table 2), facilitating maintenance of adequate water supply. The vertically developing root structure is considered to be a typical root structure that facilitates drought tolerance (Current Opinion in biotechnology, Rogers and Benfey, 2015). In summary,OsABA8ox2Overexpression inhibits the elongation growth and development of rice root systems, and knockout can optimize the rice root system structure to strengthen drought tolerance.

TABLE 2 control and drought group root parameters

The different lower case letters following the same column of values indicate significant differences by Duncan's multiple range test ((P < 0.05）。

Example 6

OsABA8ox2Knock-out and over-expression rice strain soil drought stress treatment and drought tolerance analysis

The soil drought tolerance analysis was performed by stopping watering of the 5-leaf stage rice plants. After the watering was stopped for 4 days, the water was removed,OsABA8ox2Overexpression of transgenic line OE1 and OE2 initially exhibited a rolling leaf phenotype, while WT and knock-out lines KO1 and KO2 leaves remained flat (fig. 7B). After 5 days of stopped watering, severe wilting phenotype occurred with OE1 and OE2, the WT plants also showed leaf rolling, while approximately half of the leaves remained flat with KO1 and KO2 (fig. 7C). After 7 days of soil drought treatment, rehydration treatment (recovery of watering) was performed and all KO plants recovered vigor (fig. 7D). The survival rates of OE1 and OE2 were much lower than wild type, whereas KO strains were 100% and much higher than wild type (fig. 7E). Analysis of soil drought stress tolerance indicates that, compared to wild type,OsABA8ox2the drought tolerance of the knockout strain is greatly improved, and the overexpression strain is highly sensitive to soil drought stress.

Example 7

OsABA8oxPromoter and GUS fusion expression vector pCAMBIA1303-PRO _OsABA8ox :GUSConstruction of (3) and analysis of GUS staining of transgenic lines

To proceed toOsABA8ox2Promoter analysis, total 1644bp sequence of 1619 bp (SEQ ID No: 5) upstream of ATG initiation codon and 25 bp at 5' end of coding sequence, and primer pairOsABA8ox2-Pro(Table 1), PCR amplification was performed using Kitaake genomic DNA as a template. To proceed toOsABA8ox1promoter analysis, total 973 bp sequence of 894 bp (SEQ ID No: 9) upstream of ATG initiation codon and 79 bp at 5' end of coding sequence, primer pairOsABA8ox1-Pro(Table 1) PCR amplification was performed. To proceed toOsABA8ox3Promoter analysis, total 1616 bp sequence of 1417 bp (SEQ ID No: 10) upstream of ATG initiation codon and 199 bp at 5' end of coding sequence, and primer pairOsABA8ox3-Pro(Table 1) PCR amplification was performed. Integrating the amplified fragments verified by sequencing into a primer by a homologous recombination methodHindIII (NEB) andNco (NEB) double digested pCAMBIA1303 vector, GUS was subjected toOsABA8ox1，OsABA8ox2AndOsABA8ox3Is driven by the promoter of (1). The GBclonart Seamless Cloning Kit was selected for homologous recombination. Using Agrobacterium-mediated transformation, japonica rice cultivar Kitaake was obtained as background in the same manner as in example 1PRO _OsABA8ox :GUSA transgenic line.

GUS staining kit (Coolaber, Beijing) was selected for GUS staining.PRO _OsABA8ox :GUSGUS staining of transgenic rice line shows that the transgenic rice line is in vegetative growth periodOsABA8ox2Mainly in the root (FIG. 8B) and strongly in the radicle meristematic region (FIG. 9A).OsABA8ox3Mainly expressed in leaves (FIG. 8A). WhileOsABA8ox1The expression was very weak at the seedling stage and almost absent (FIG. 8). The expression patterns of these 3 genes are shown to be space-time specific, suggesting that their promoters have several different cis-acting elements.OsABA8ox2Stronger expression was seen in both glume flowers and seeds at the filling stage (fig. 8C, fig. 9B).

Sequence listing

<110> institute of biotechnology of Chinese academy of agricultural sciences

Application of <120> ABA 8' -hydroxylase gene OsABA8ox2 in plant photomorphogenesis and root development

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 510

<212> PRT

<213> Rice (Oryza sativa)

<400> 1

Met Ala Phe Leu Leu Phe Phe Val Phe Val Thr Ala Ala Val Leu Cys

1 5 10 15

Phe Val Val Pro Ala Phe Leu Leu Leu Cys Thr Ser Val Gln Arg Arg

20 25 30

Arg Asp Val Gly Gln Gly Gly Gly Arg Asp Trp Gln Lys Lys Lys Lys

35 40 45

Leu Arg Leu Pro Pro Gly Ser Met Gly Trp Pro Tyr Val Gly Glu Thr

50 55 60

Leu Gln Leu Tyr Ser Gln Asp Pro Asn Val Phe Phe Ala Ser Lys Gln

65 70 75 80

Lys Arg Tyr Gly Glu Ile Phe Lys Thr Asn Leu Leu Gly Cys Pro Cys

85 90 95

Val Met Leu Ala Ser Pro Glu Ala Ala Arg Phe Val Leu Val Ser Gln

100 105 110

Ala Arg Leu Phe Lys Pro Thr Tyr Pro Pro Ser Lys Glu Arg Met Ile

115 120 125

Gly Pro Ser Ala Leu Phe Phe His Gln Gly Glu Tyr His Leu Arg Leu

130 135 140

Arg Arg Leu Val Gln Ala Ala Leu Ala Pro Asp Ser Leu Arg Ala Leu

145 150 155 160

Val Pro Asp Val Asp Ala Ala Val Ala Ala Thr Leu Ala Ala Trp Ser

165 170 175

Gly Gly His Val Ala Ser Thr Phe His Ala Met Lys Lys Leu Ser Phe

180 185 190

Asp Val Gly Val Val Thr Ile Phe Gly Gly Arg Leu Gly Arg Arg His

195 200 205

Arg Glu Glu Leu Arg Thr Asn Tyr Ser Val Val Glu Arg Gly Tyr Asn

210 215 220

Cys Phe Pro Asn Arg Phe Pro Gly Thr Leu Tyr His Lys Ala Ile Gln

225 230 235 240

Ala Arg Lys Arg Leu Arg Ala Ile Leu Ser Glu Ile Val Ala Glu Arg

245 250 255

Arg Ala Arg Gly Gly Gly Gly Gly Gly Gly Gly Asp Asp Leu Leu Gly

260 265 270

Gly Leu Met Arg Ser Arg Asp Asp Gly Thr Ala Gly Ala Val Ala Leu

275 280 285

Leu Thr Asp Asp Gln Ile Ala Asp Asn Val Val Gly Val Leu Phe Ala

290 295 300

Ala Gln Asp Thr Thr Ala Ser Val Leu Thr Trp Ile Leu Lys Tyr Leu

305 310 315 320

His Asp Ser Pro Lys Leu Leu Glu Ala Val Lys Ala Glu Gln Met Ala

325 330 335

Ile Tyr Val Ala Asn Glu Gly Gly Lys Arg Pro Leu Thr Trp Thr Gln

340 345 350

Thr Arg Ser Met Thr Leu Thr His Gln Val Ile Leu Glu Ser Leu Arg

355 360 365

Met Ala Ser Ile Ile Ser Phe Thr Phe Arg Glu Ala Val Ala Asp Val

370 375 380

Glu Tyr Lys Gly Phe Leu Ile Pro Lys Gly Trp Lys Val Met Pro Leu

385 390 395 400

Phe Arg Asn Ile His His Asn Pro Asp Tyr Phe Gln Asp Pro Gln Lys

405 410 415

Phe Asp Pro Ser Arg Phe Lys Val Ala Pro Arg Pro Ser Thr Phe Leu

420 425 430

Pro Phe Gly Ser Gly Val His Ala Cys Pro Gly Asn Glu Leu Ala Lys

435 440 445

Leu Glu Met Leu Val Leu Val His Arg Leu Val Thr Ala Tyr Arg Trp

450 455 460

Glu Ile Val Gly Ala Ser Asp Glu Val Glu Tyr Ser Pro Phe Pro Val

465 470 475 480

Pro Arg Gly Gly Leu Asn Ala Lys Leu Trp Lys Gln Glu Ala Glu Glu

485 490 495

Asp Met Tyr Met Ala Met Gly Thr Ile Thr Ala Ala Gly Ala

500 505 510

<210> 2

<211> 417

<212> PRT

<213> Rice (Oryza sativa)

<400> 2

Met Ala Phe Leu Leu Phe Phe Val Phe Val Thr Ala Ala Val Leu Cys

1 5 10 15

Phe Val Val Pro Ala Phe Leu Leu Leu Cys Thr Ser Val Gln Arg Arg

20 25 30

Arg Asp Val Gly Thr Gly Trp Arg Ala Arg Leu Ala Glu Glu Glu Glu

35 40 45

Ala Gln Ala Ser Ser Gly Ile His Gly Leu Ala Val Arg Arg Arg Asp

50 55 60

Ala Pro Ala Leu Leu Pro Gly Pro Gln Arg Leu Leu Arg Leu Gln Ala

65 70 75 80

Glu Glu Val Arg Arg Asp Ile Gln Asp Glu Ser Ala Gly Val Pro Val

85 90 95

Arg Asp Ala Gly Glu Pro Gly Gly Gly Glu Val Arg Ala Gly Val Ala

100 105 110

Gly Glu Ala Val Gln Ala Asp Val Pro Ala Glu Gln Gly Ala Asp Asp

115 120 125

Arg Ala Val Gly Ala Leu Leu Pro Pro Gly Arg Val Pro Pro Pro Pro

130 135 140

Pro Pro Pro Arg Pro Gly Arg Pro Arg Pro Gly Leu Pro Pro Arg Pro

145 150 155 160

Arg Pro Gly Arg Arg Arg Arg Arg Arg Arg His Ala Arg Arg Leu Val

165 170 175

Arg Arg Pro Arg Arg Gln His Leu Pro Arg His Glu Glu Ala Leu Val

180 185 190

Arg Arg Arg Arg Arg Asp His Leu Arg Arg Pro Ala Arg Pro Pro Ala

195 200 205

Gln Gly Gly Ala Glu Asp Glu Leu Leu Arg Arg Gly Glu Arg Leu Gln

210 215 220

Leu Leu Pro Gln Pro Leu Pro Gly Asp Ala Leu Pro Gln Gly Asp Pro

225 230 235 240

Gly Glu Glu Ala Ala Ala Arg Asp Pro Glu Arg Asp Arg Gly Gly Ala

245 250 255

Ala Gly Ala Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Pro Pro Arg

260 265 270

Arg Pro His Ala Val Ala Arg Arg Arg His Arg Arg Arg Gly Gly Ala

275 280 285

Ala His Arg Arg Pro Asp Arg Arg Gln Arg Arg Arg Arg Ala Val Arg

290 295 300

Gly Ala Gly His His Arg Gln Arg Pro His Leu Asp Pro Gln Val Pro

305 310 315 320

Pro Arg Leu Ala Glu Ala Ser Arg Ser Arg Gln Gly Gly Ala Asp Gly

325 330 335

Asp Leu Arg Gly Gln Arg Gly Arg Glu Ala Ala Ala Asp Val Asp Ala

340 345 350

Asp Glu Glu His Asp Thr His Ala Ser Gly Tyr Thr Gly Glu Leu Glu

355 360 365

Asp Gly Glu His Asn Leu Leu His Val Gln Arg Gly Ser Arg Arg Arg

370 375 380

Gly Val Gln Arg Phe Pro Asp Ser Lys Gly Val Glu Gly Asp Ala Ser

385 390 395 400

Val Gln Glu His Pro Ser Gln Pro Gly Leu Leu Pro Gly Ser Thr Lys

405 410 415

Val

<210> 3

<211> 416

<212> PRT

<213> Rice (Oryza sativa)

<400> 3

Met Ala Phe Leu Leu Phe Phe Val Phe Val Thr Ala Ala Val Leu Cys

1 5 10 15

Phe Val Val Pro Ala Phe Leu Leu Leu Cys Thr Ser Val Gln Arg Arg

20 25 30

Arg Asp Val Thr Gly Trp Arg Ala Arg Leu Ala Glu Glu Glu Glu Ala

35 40 45

Gln Ala Ser Ser Gly Ile His Gly Leu Ala Val Arg Arg Arg Asp Ala

50 55 60

Pro Ala Leu Leu Pro Gly Pro Gln Arg Leu Leu Arg Leu Gln Ala Glu

65 70 75 80

Glu Val Arg Arg Asp Ile Gln Asp Glu Ser Ala Gly Val Pro Val Arg

85 90 95

Asp Ala Gly Glu Pro Gly Gly Gly Glu Val Arg Ala Gly Val Ala Gly

100 105 110

Glu Ala Val Gln Ala Asp Val Pro Ala Glu Gln Gly Ala Asp Asp Arg

115 120 125

Ala Val Gly Ala Leu Leu Pro Pro Gly Arg Val Pro Pro Pro Pro Pro

130 135 140

Pro Pro Arg Pro Gly Arg Pro Arg Pro Gly Leu Pro Pro Arg Pro Arg

145 150 155 160

Pro Gly Arg Arg Arg Arg Arg Arg Arg His Ala Arg Arg Leu Val Arg

165 170 175

Arg Pro Arg Arg Gln His Leu Pro Arg His Glu Glu Ala Leu Val Arg

180 185 190

Arg Arg Arg Arg Asp His Leu Arg Arg Pro Ala Arg Pro Pro Ala Gln

195 200 205

Gly Gly Ala Glu Asp Glu Leu Leu Arg Arg Gly Glu Arg Leu Gln Leu

210 215 220

Leu Pro Gln Pro Leu Pro Gly Asp Ala Leu Pro Gln Gly Asp Pro Gly

225 230 235 240

Glu Glu Ala Ala Ala Arg Asp Pro Glu Arg Asp Arg Gly Gly Ala Ala

245 250 255

Gly Ala Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Pro Pro Arg Arg

260 265 270

Pro His Ala Val Ala Arg Arg Arg His Arg Arg Arg Gly Gly Ala Ala

275 280 285

His Arg Arg Pro Asp Arg Arg Gln Arg Arg Arg Arg Ala Val Arg Gly

290 295 300

Ala Gly His His Arg Gln Arg Pro His Leu Asp Pro Gln Val Pro Pro

305 310 315 320

Arg Leu Ala Glu Ala Ser Arg Ser Arg Gln Gly Gly Ala Asp Gly Asp

325 330 335

Leu Arg Gly Gln Arg Gly Arg Glu Ala Ala Ala Asp Val Asp Ala Asp

340 345 350

Glu Glu His Asp Thr His Ala Ser Gly Tyr Thr Gly Glu Leu Glu Asp

355 360 365

Gly Glu His Asn Leu Leu His Val Gln Arg Gly Ser Arg Arg Arg Gly

370 375 380

Val Gln Arg Phe Pro Asp Ser Lys Gly Val Glu Gly Asp Ala Ser Val

385 390 395 400

Gln Glu His Pro Ser Gln Pro Gly Leu Leu Pro Gly Ser Thr Lys Val

405 410 415

<210> 4

<211> 1533

<212> DNA

<213> Rice (Oryza sativa)

<400> 4

atggctttct tgctcttctt tgtctttgtg acagctgcag tgctgtgctt cgtcgtcccg 60

gcgttcttgc tgctctgcac gagcgtgcag aggaggagag atgttggaca gggtggaggg 120

cgagattggc agaagaagaa gaagctcagg cttcctccgg gatccatggg ctggccgtac 180

gtcggcgaga cgctccagct ctactcccag gaccccaacg tcttcttcgc ctccaagcag 240

aagaggtacg gcgagatatt caagacgaat ctgctggggt gcccgtgcgt gatgctggcg 300

agcccggagg cggcgaggtt cgtgctggtg tcgcaggcga ggctgttcaa gccgacgtac 360

ccgccgagca aggagcggat gatcgggccg tcggcgctct tcttccacca gggcgagtac 420

cacctccgcc tccgccgcct cgtccaggcc gccctcgccc cggactccct ccgcgccctc 480

gtcccggacg tcgacgccgc cgtcgccgcc acgctcgccg cctggtccgg cggccacgtc 540

gccagcacct tccacgccat gaagaagctc tcgttcgacg tcggcgtcgt gaccatcttc 600

ggcggccggc tcggccgccg gcacagggag gagctgagga cgaactactc cgtcgtggag 660

agaggctaca actgcttccc caaccgcttc ccggggacgc tctaccacaa ggcgatccag 720

gcgaggaagc ggctgcgcgc gatcctgagc gagatcgtgg cggagcggcg ggcgcgcggc 780

ggcggcggcg gcggcggcgg cgacgacctc ctcggcggcc tcatgcggtc gcgcgacgac 840

ggcaccgccg gcgcggtggc gctgctcacc gacgaccaga tcgccgacaa cgtcgtcggc 900

gtgctgttcg cggcgcagga caccaccgcc agcgtcctca cctggatcct caagtacctc 960

cacgactcgc cgaagcttct cgaagccgtc aaggcggagc agatggcgat ctacgtggcc 1020

aacgagggcg ggaagcggcc gctgacgtgg acgcagacga ggagcatgac actcacgcat 1080

caggttatac tggagagctt gaggatggcg agcataatct ccttcacgtt cagagaggca 1140

gtcgccgacg tggagtacaa aggtttcctg attccaaagg ggtggaaggt gatgcctctg 1200

ttcaggaaca tccatcacaa cccggactac ttccaggatc cacaaaagtt tgatccttct 1260

agattcaagg tggcgccgcg tccaagcacg ttcctgccgt tcgggagcgg cgtgcacgcg 1320

tgcccgggca acgagctggc caagctggag atgctcgtcc tcgtccaccg cctcgtcacc 1380

gcctacaggt gggagatcgt cggggcgagc gacgaggtgg agtacagccc gttcccggtg 1440

ccgaggggcg ggctcaacgc caagctgtgg aagcaggagg cggaggagga catgtacatg 1500

gccatgggca ccatcacagc agcaggtgct tga 1533

<210> 5

<211> 1619

<212> DNA

<213> Rice (Oryza sativa)

<400> 5

aaacaagaac aaatactcca atatttgcta gattgggtat aggcccgttg atgcattcat 60

cgaattattg ggaattccat ctcaaaactc acaaaatctt atataaaaca ttgcaaaaaa 120

aaaacacttc tattgcaaaa atcttgcaga taaattttct ttttccaatg atgcagctta 180

tatttatcac ttaaaaaagt catctcctgt gccgtggctc catttgctcc cttttattta 240

tttcccccca tcccggttat agatcaaatc catttgacca ttaaaagaag ccaaaataat 300

caagcagtac cagatcacac aaggcatcta gaaagaaaga agaaaaaaaa cgatattttt 360

tttaaaagaa tcaggaaaaa tatatccagg aaccatggta caaactactg aaactgttgt 420

gcaaacttct tgatcttttg ggatatttct aatttaggtt tttttttgtt gaattaactg 480

aacagtggac accttttttt tcaaaaaaaa tctttgacca aatgagaata gtttaaaaga 540

aatgaagaga gctgtgacaa aagagagtac cgtcttcatg cggaagaaat ggcattagag 600

gttatacaag tggcaagtaa aaatggtcta aactttatgg caggctagta aaaaatagta 660

atataaacaa caacagtcta agcaaaattt aactaaggat tacaacaatc cataatgatg 720

tagtaccaat caagctgtca gagagatctc aggctctgat gctatgttga atctcaatcc 780

ccaaatcaat gcctgtccat gggccaaatc ccagggaatt cgtgcaagcc ccgctgagct 840

gcacatgact aggaccctat ttgaaagatt agcccagaac taattttgag agctaatatt 900

tagtaatgaa ttggtaggct aaacattagt ctaggttggt atgtttggat ctatgggcta 960

attcaaggct aaaaggtaga gagagagtag aaagagagga gagagaagga gagagagagc 1020

tgctttttga tggtccccac acaaaattag ccccattagc acttcttaga gaggctaata 1080

ttttgagagg ggctaattca tattagctca aaattagaca acctgtttgg attcttgaga 1140

gctaatttaa ggctaaaagg taagagctaa acattagccc atggaaacaa acagggccag 1200

gaatcttcta atctcccttt catgtgtccc tccttttttt ctccctatcc ccaaagattt 1260

cacattttct aatgagcatc atgccgtcca tgagctctgt tttttatcct aacacccctc 1320

tgcagccgtg ctcaccctct ataaataccc catacaggtg tcttccaata gcaccaccat 1380

ttggaagaat cctccccaag attgctcgat cccctcgtgt ccctctctcc caacaacact 1440

gcgccacaga cacaaatcac actcagatac gaaagataag tacagagaga gagagagggc 1500

acggattata cactgcacac aagcatatat atatatatat cgattagcca tccgtgctga 1560

tctgaagagt atcatcggta gagagtttta cagagttgtt tggacaagga gacacacac 1619

<210> 6

<211> 19

<212> DNA

<213> Rice (Oryza sativa)

<400> 6

ggaggagaga tgttggaca 19

<210> 7

<211> 20

<212> DNA

<213> Rice (Oryza sativa)

<400> 7

ggaggagaga tgttgggaca 20

<210> 8

<211> 17

<212> DNA

<213> Rice (Oryza sativa)

<400> 8

ggaggagaga tgtgaca 17

<210> 9

<211> 894

<212> DNA

<213> Rice (Oryza sativa)

<400> 9

cagtatgatg gctggttggg gatgatcaga ggaagcggcc aaaggagagc agcattatag 60

gagcaggagc aggagcagga gcagcagcag ctgctgccgg ggcagtacgt agtacgtacg 120

agagcgcgta cagtagctga tggtgtggaa acagggcgga aagggagagg tgagctggag 180

aaagggatga ttctctcgcg cgctggccag aagagaagag aggcccattt agctatcact 240

gtattggtta accggtttaa ccgatcagtt gattaatccg gctctctcgc ccgcacgcct 300

ttttcccttt atttccgtat tgattgcgca attattcggc agcgtctatc tgtctctctc 360

gcgcgcggag gcacccagct cggctattct agcgcgtagt ggtggtgcta gctactagta 420

gtagtagatt tttgggcagc aagcggccgc agaaaaggag agcgaaacgg aggagaaacc 480

gggaaactca gctcgcggcc aatctataaa tagctgccac ccctctcgcc tttccctcca 540

cacccccccc aacaacacca ccgcccattt ccctttctct tcctcttctt cttccttctc 600

ctcctactcc tctgcgattg acaaagataa gtgaagtgag caggcgccaa tgggtgcttt 660

tcttctgttc gtgtgcgtgc tcgcgccttt cttgcttgtc tgcgccgtcc gcggccgccg 720

ccggcaggcg ggctcgtcgg aagcggcggc gtgcggcctg ccgctgccgc cggggtcgat 780

ggggtggccg tacgtcgggg agacgttcca gctgtactcg tccaagaacc ccaacgtgtt 840

cttcaacaag aagcggaaca agtacggtcc catcttcaag acgcacatcc tggg 894

<210> 10

<211> 1417

<212> DNA

<213> Rice (Oryza sativa)

<400> 10

agttgctgtt ggtcattttg gtttagcccc gctatatttt tatcctggct ccgccactgt 60

attctagtag tcctagtatg tcctatctct atgtatactt ttagatgatg tggcacatac 120

ttaaagtata agtaggagta aactatactg tattaaacta cattttaata tgtacagaac 180

gacgataaaa ctggataagt tgaacttatc ttgctgcatg tacgtttcat ccccaaaaaa 240

cacatgataa gaacataata aatgcaatgc aatgctattc ttacgaccag attggccaat 300

acacgattga aactaatcag cacagggtag caaatcaaca gggtatagtg gtatactgaa 360

cttatcttgc tgcatgtacg tttcaccctc ttaagaaaat cttgataaga aataaatacc 420

acgatatata tatgctatca ttagaaatcc acaggataag ttgaaacttc cttgatgtat 480

gtacatttag gcgctgcttc tttcagctta ggattattat aatccaaatt attaggagta 540

aactgaaaga aacaaacaac ttattgaagt agcttattat aatctggagc ctagcttagg 600

ccctctttat ttaggcttag gtttattagc ctagactttt aagtcagctt atatgattta 660

taagccggtg gatttaatgt cctaagttta gtagttgagt catacatcta actcacataa 720

gccaaaaaag cttctccaac ctagcttttg gcttaatagt gttagagtgg cttatggctt 780

caaaaaaacc aaacggaaaa gctgcttgct tgtttaggct cagacttttc gacttataag 840

ttggcttata agcctaaaca aagagggctt attataatat gataagctca tttaggtgag 900

ctttttccag attattgggt aaaaaattac ccgtcatacc acaccacttt ctctttagac 960

ttgtaaaccc aataatttag gatgtaataa tctaggaaag aaacaactaa ccgtttattc 1020

tactacagat tataacaatc tagcttatgg taatctgact caataatcta gattataata 1080

atcctaagct gggaaaaaca ggccttactc cctcccctcc ctagctcgcg gacaccatat 1140

gagcttccaa actggcctct cactttctct ctcgcgctct gcctctataa ataccacgca 1200

aggccttccc acgcccacca ccattcgaaa gatccctcga agatttttct cctccctctc 1260

tccctctcct cacagctctc tccctctctc ctagcaacag catatatagc gcaaagacaa 1320

taacacacga ctagctagca agaagaagac agctggttca gaactctcaa aacagctgtg 1380

gtgtccaagt gatccttaat tctccagggt catttgg 1417

Claims (9)

1. ABA 8' -hydroxylase GeneOsABA8ox2The application in plant photomorphogenesis and root development is characterized by that it uses the inactivation of ABA 8' -hydroxylase gene in plant bodyOsABA8ox2The plant photomorphogenesis is positively regulated and controlled, the shade-avoiding reaction is relieved, the shade tolerance is improved, and the root system structure is optimized.
2. 2. ABA 8' -hydroxylase gene according to claim 1OsABA8ox2the application in plant photomorphogenesis and root development is characterized in that the inactivation technology is a gene editing technology; the edited gene isOsABA8ox2The amino acid sequence of the encoded protein is shown in a sequence table SEQ ID No: 1 is shown in the specification; the plant is rice of Gramineae.
3. 3. ABA 8' -hydroxylase gene according to claim 2OsABA8ox2The application in plant photomorphogenesis and root development is characterized in that the target sequence edited by the gene is shown in a sequence table SEQ ID No: 6, and the mutant sequence is shown in a sequence table SEQ ID No: 7 and SEQ ID No: 8 is shown in the specification; the mutant amino acid sequence is shown in a sequence table SEQ ID No: 2 and SEQ Id No: 3, respectively.
4. 4.OsABA8ox2The promoter is used for expressing and applying in root meristems, glume flowers and seeds in the filling stage.
5. 5. The method of claim 4OsABA8ox2The expression application of the promoter in root meristems, glume flowers and seeds in the filling stage is characterized in that the promoter is used for expressing the root meristems, the glume flowers and the seeds in the filling stageOsABA8ox2The nucleotide sequence of the promoter is shown in a sequence table SEQ ID No: 5, respectively.
6. 6.OsABA8ox2The coding gene and the application of the mutant gene in regulating and controlling plant photomorphogenesis and root development.
7. 7. the method of claim 6OsABA8ox2The coding gene and the application of the mutant gene in the regulation of plant photomorphogenesis and root development are characterized in thatOsABA8ox2The nucleotide sequence of the coding gene is shown in a sequence table SEQ ID No: 4, the target sequence edited by the gene is shown as a sequence table SEQ ID No: 6, the mutant sequence is shown in a sequence table SEQ ID No: 7 and SEQ ID No: shown in fig. 8.
8. 8. The method of claim 6OsABA8ox2The coding gene and the application of the mutant gene in the regulation of plant photomorphogenesis and root development are characterized in thatOsABA8ox2the sequence of the protein product expressed by the coding gene is shown in a sequence table SEQ ID No: 1, and the mutant sequence is shown in a sequence table SEQ ID No: 2 and SEQ ID No: 3, respectively.
9. 9. The method of claims 6-8OsABA8ox2The coding gene and the application of the mutant gene in the regulation of plant photomorphogenesis and root development are characterized in thatOsABA8ox2The complete or partial DNA of the coding gene is constructed into a recombinant vector or an expression cassette and transferred into a cell line or engineering bacteria.