Application of PAA4 gene in regulation and control of rice panicle development

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a PAA4 gene in regulation and control of rice panicle development. The invention discloses a PPA4 gene and application of protein coded by the PPA4 gene in regulation and control of rice panicle development. The amino acid sequence of the protein coded by the PPA4 gene is shown as SEQ ID No.2, and the nucleotide sequence of a coding region of the PPA4 gene is shown as SEQ ID No.1. The invention further discloses a preparation method of the protein coded by the PPA4 gene and application of the protein coded by the PPA4 gene in regulation and control of rice panicle development. The PAA4 gene participates in cell detoxification and antioxidant pathways, plays a key role in regulating active oxygen in spikes, regulates the development of spike tips by influencing the balance of rice spike tip active oxygen (ROS), can be applied to rice spike character breeding, provides spike degeneration resistant gene resources for production and application, and has wide application prospects in rice molecular breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of the PAA4 gene in regulating the development of rice panicles. Background Art

[0002] Rice is the most important food crop in the world, and its yield is affected by the number of effective panicles, the number of grains per panicle, and the thousand-grain weight. The number of effective panicles is determined by the number of tillers, while the number of grains per panicle and the thousand-grain weight are closely related to panicle development (Xing et al., 2010). Rice panicle development involves the differentiation of multiple organs and complex regulatory mechanisms. First, the stem apex meristem is stimulated by the flowering signal and differentiates into the inflorescence meristem, and then the inflorescence meristem differentiates into the primary stalk meristem and the main panicle axis. The main panicle axis continues to differentiate into the primary stalk meristem, and stops differentiating after reaching a certain length. Thereafter, the primary stalk meristem differentiates into the secondary stalk meristem and the spikelet meristem, and finally forms a complete stalk and spikelet (Komatsu et al., 2003; Wang et al., 2005). At present, many genes involved in the development of stalks and spikelets have been cloned and reported. For example, LAX1 interacts with LAX2 to regulate axillary bud meristem formation in rice (Komatsu et al., 2001; Komatsu et al., 2003; Tabuchi et al., 2011). FZP is a transcription factor that regulates the expression of OsMADS genes, which then determines the characteristics of floral organs (Bai et al., 2016). The E3 ubiquitin ligase OsUPL2 controls panicle size and panicle number by interacting with APO1 and APO2 and negatively regulating their stability (Huang et al., 2021; Ookawa et al., 2010).

[0003] Rice panicle degeneration can significantly affect yield. Previous studies have mainly focused on collecting panicle degeneration phenotypes, and little research has been done on its mechanism. The panicle apex degeneration of paab1-1 is due to the imbalance of reactive oxygen species in the panicle apex caused by the disruption of the function of the malate transporter it encodes, which leads to programmed cell death (Heng et al., 2018). The panicle degeneration of paa1019 and dps1 is also programmed cell death caused by the imbalance of reactive oxygen species, among which PAA1019 encodes a calcineurin B-like subunit-interacting protein kinase, and DPS1 encodes a protein containing a cystathionine β-synthase domain that is essential for the development of anther epidermis and panicle (Peng et al., 2018, Zafar et al., 2020). SPL6 is highly expressed at the spike tip, which inhibits the transcription of the endoplasmic reticulum stress sensor IRE1 and controls the output intensity of the stress signal. Loss of SPL6 function leads to overexpression of IRE1, resulting in uncontrolled output of endoplasmic reticulum stress signals and excessive expression of downstream genes, leading to senescence and degeneration of spikelet cells at the top (Wang et al., 2018). In addition, TUTOU1 encodes a cAMP-like receptor protein inhibitor that is essential for actin assembly. Mutations in TUTOU1 can lead to spike tip degeneration and defects in anther and pollen development (Bai et al., 2015).

[0004] The mitogen-activated protein kinase (MAPK) cascade pathway is a highly conserved signaling pathway present in eukaryotic cells. Current studies have shown that the MAPK cascade in rice is associated with important physiological processes such as plant immunity, response to abiotic stress, environmental signals, hormone production and response, and grain size regulation (Zhang et al., 2022). In recent years, researchers have also found that the MAPK cascade plays a role in regulating the number of grains per panicle in rice. The OsMKKK10-OsMKK4-OsMPK6 cascade binds to its downstream GSN1 to coordinate the balance between the number of grains per panicle and grain size in rice (Guo et al., 2018).

[0005] Although abiotic stress is one of the main factors inducing rice panicle degeneration, it has been found in recent years that panicle degeneration is also regulated by intrinsic genes. Currently, research on rice panicle degeneration is still focused on material collection and phenotypic identification, and the core mechanism has not yet been explored. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides an application of the PPA4 gene in regulating the development of rice panicles. The function of the PPA4 gene in regulating the development of rice panicles has not been reported in the prior art. Therefore, the study of the function of the PPA4 gene in regulating the development of rice panicles will enrich the regulatory network of rice panicle development and provide gene resources for studying the regulatory mechanism of rice panicle development.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] The present invention provides application of the PAA4 gene in regulating rice panicle development. The amino acid sequence of the protein encoded by the PAA4 gene is shown in SEQ ID No.2.

[0009] Furthermore, the nucleotide sequence of the PAA4 gene coding region is shown in SEQ ID No.1.

[0010] Furthermore, the application is the application of the PAA4 gene in regulating rice length, panicle length, number of branches and stalks, and number of grains per panicle.

[0011] The present invention also provides the use of a protein encoded by the PAA4 gene in regulating the development of rice panicles. The amino acid sequence of the protein encoded by the PAA4 gene is shown in SEQ ID No.2.

[0012] Furthermore, the application is the use of the protein encoded by the PAA4 gene in regulating rice length, panicle length, number of branches and stalks, and number of grains per panicle.

[0013] The present invention also provides the use of the PAA4 gene in the breeding of rice panicle traits. The amino acid sequence of the protein encoded by the PAA4 gene is shown in SEQ ID No.2.

[0014] Furthermore, the nucleotide sequence of the PAA4 gene coding region is shown in SEQ ID No.1.

[0015] The present invention also provides the use of a protein encoded by the PAA4 gene in rice panicle trait selection and breeding. The amino acid sequence of the protein encoded by the PAA4 gene is shown in SEQ ID No.2.

[0016] The invention process of the present invention is as follows: the present invention identified a rice panicle tip degeneration mutant (named paa4) from the EMS mutagenesis library of the rice indica maintenance line Xida 1B. Compared with the wild type (WT), the mutant paa4 plants became shorter, the panicle tip showed severe degeneration, and the degeneration rate reached about 25%. Secondly, the top florets of the paa4 panicle began to show developmental retardation when the length of the panicle was 7 cm, and physiological indicators and experimental data showed that the paa4 panicle tip showed an imbalance of active oxygen at this time, which eventually led to programmed cell death.

[0017] Genetic analysis confirmed that the mutant was controlled by a recessive single gene (named PAA4). Positional cloning was performed and PAA4 was finally located between D4C-3 and D4C-4, with a physical distance of 75kb. Sequencing analysis of this localization interval revealed that there was a base substitution from G (SEQ ID No.1) to A (SEQ ID No.3) in the 19th exon of the gene in LOC_Os04g56530. The mutant gene contains a base substitution, resulting in the encoded glycine (G) (SEQ ID No.2) becoming aspartic acid (D) (SEQ ID No.4).

[0018] The expression pattern of PAA4 was studied and it was found that PAA4 has a constitutive expression pattern. In addition, the expression level in the husk is much higher than that in organs such as pistil.

[0019] Transcriptome sequencing analysis of the spike tips of the wild type and paa4 showed that PAA4 is involved in cellular detoxification and antioxidant pathways and plays a key role in regulating reactive oxygen species in the spike.

[0020] The above results indicate that the PAA4 gene is involved in the cellular detoxification and antioxidant pathways, plays a key role in regulating reactive oxygen in the panicle, and regulates the development of the panicle tip by affecting the balance of reactive oxygen species (ROS) in the rice panicle tip. It can be used to regulate the development of rice panicles and the breeding of rice panicle traits, providing genetic resources for resistance to panicle degeneration for production applications.

[0021] The beneficial effects of the present invention are as follows: the present invention provides the application of the PPA4 gene and the protein encoded by the PPA4 gene in regulating the development of rice panicle, the amino acid sequence of the protein encoded by the PPA4 gene is shown in SEQ ID No. 2, and the nucleotide sequence of the coding region of the PPA4 gene is shown in SEQ ID No. 1. The PAA4 gene participates in the cell detoxification and antioxidant pathways, plays a key role in regulating reactive oxygen in the panicle, regulates the development of the panicle tip by affecting the balance of reactive oxygen (ROS) in the panicle tip of rice, can be applied to the breeding of rice panicle traits, provides gene resources for anti-panicle degeneration for production applications, and has broad application prospects in rice molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 for phenotypic identification; A is the whole plant phenotype of mature plants; B is the ear phenotype at heading stage; C is the ear phenotype at maturity; D and E are enlarged views of the box in B; F and G are enlarged views of the box in C; H is a comparison of plant height between the wild type and paa4 at maturity; I is a comparison of ear length between the wild type and paa4 at maturity; J is a comparison of ear degeneration rates between the wild type and paa4 at maturity; K is a comparison of grain number per ear; L is a comparison of fruit set rate; M is a comparison of thousand-grain weight.

[0023] Figure 2For phenotypic identification; A is the expansion of the panicle branches of the wild type and paa4; B is the wild type pollen; C is the paa4 pollen; D is the primary branch statistics; E is the secondary branch statistics; F is the pollen fertility statistics.

[0024] Figure 3 For ear phenotype identification; A to I are ears of different lengths, and A1 to I2 are enlarged views of A to I respectively.

[0025] Figure 4 For the determination of physiological indicators; A is the apical husk of ears of different lengths; B1 to J2 are trypan blue, DAB and NBT staining, K is the determination of hydrogen peroxide content, and L is the determination of malondialdehyde content.

[0026] Figure 5 For the detection of programmed cell death; A to I2 are TUNEL experiments; J to M are RT-PCR results of programmed cell death and reactive oxygen species related genes.

[0027] Figure 6 is map-based cloning; A is the gene localization map; B is the PAA4 gene structure; C to F are the complementary plant phenotypes; G is the complementary sequencing result; H is the ear degeneration rate of the complementary plant.

[0028] Figure 7 Protein structure analysis; A is the PAA4 protein structure; B is the PAA4 evolutionary tree; C is the mutation site alignment; D is the PAA4 protein tertiary structure prediction.

[0029] Figure 8 A is the expression pattern; A is the expression level of PAA4 in each tissue; B to I are GUS staining in each tissue; J to M are subcellular localization.

[0030] Fig. 9 is the transcriptome data; A is the enrichment of cell detoxification pathway; B is the heat map of cell detoxification pathway genes; CJ is the expression level of the corresponding gene.

[0031] All wild-types in the figures are indicated by WT. DETAILED DESCRIPTION

[0032] The present invention is described clearly and completely below through specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention belong to the protection scope of the present invention.

[0033] Example 1. Gene screening and cloning

[0034] (1) Discovery of mutant paa4

[0035] The inventors identified a panicle apex degeneration mutant paa4 in the EMS mutagenesis library of the indica rice maintainer line Xida 1B. Under field planting conditions, paa4 showed a semi-dwarf phenotype from the seedling stage to the mature stage ( Figure 1 A, H). At the heading stage, the ear length of paa4 was found to be shorter than that of the wild type (WT) and was also accompanied by ear apex degeneration ( Figure 1 B, D, E, I). After maturity, the degenerated husks at the top of the paa4 panicle gradually fell off, leaving the top branch ( Figure 1 C, F, G). The ear degeneration rate of paa4 reached about 25% ( Figure 1 J). At the same time, the number of grains per ear, fruit setting rate and thousand-grain weight of paa4 were significantly reduced ( Figure 1 K, L, M). In addition, the primary and secondary branches of paa4 were significantly less than those of wild type ( Figure 2 A, D, E), but no difference in pollen fertility ( Figure 2 B, C, F).

[0036] In order to explore the period of paa4 spikelet degeneration, the inventors divided the spikelets of wild type and paa4 into 9 stages according to the developmental length from 1 cm to 17 cm ( Figure 3 A to I). The paa4 spikelets stopped growing when they reached about 17 cm. Although the wild-type spikelets continued to grow to about 25 cm, the development of the husks of the two spikelets was the same except for the degraded husks of the paa4 spikelets. Therefore, 17 cm was defined as the last stage. The inventors found that the husks of the spikelets of paa4 spikelets had no visible degraded phenotype when they were 1-5 cm long, which was no different from the wild-type spikelets ( Figure 3 A1 to C2). However, when the floret at the top of paa4 grows to about 7 cm, it shows a slow growth ( Figure 3 D1, D2), and significant slowness appeared at 9 cm ( Figure 3 E1, E2). Then the florets at the top of the paa4 spike gradually stopped developing ( Figure 3 F1 to I2).

[0037] (2) Imbalance of reactive oxygen species in the spike tip of the paa4 mutant

[0038] The results of the analysis of the husks at the top of spikelets at different developmental stages showed that the husks at the top of the paa4 spikelets differed from those of the wild type at the 7 cm stage and showed a significant developmental lag at the 9 cm stage ( Figure 4A). Since the husks at the top of the paa4 spikelets showed developmental differences at the 7cm stage, the inventors stained the husks at the top of the spikelets at the 5cm stage, where the difference was not significant, and at the 9cm stage, where the difference was obvious, with trypan blue, DAB and NBT. Trypan blue dye can enter incomplete and broken cells, thereby distinguishing normal from dead cells. The results of trypan blue staining showed that the husks at the top of the paa4 spikelets at the 5cm stage were not much different from those of the wild type, and the color was slightly darker ( Figure 4 B1, B2; At the 7cm stage, the husk at the top of the paa4 spikelet showed a strong blue color ( Figure 4 C1, C2; at the 9cm stage, the husk at the top of the paa4 spikelet still showed a strong blue color ( Figure 4 D1, D2). DAB can determine the concentration of peroxidase (POD) in cells. DAB staining results showed that the POD concentration in the husk at the top of the paa4 panicle was higher than that of the wild type at the 5cm stage, 7cm stage and 9cm stage ( Figure 4 E1 to G2). NBT can detect superoxide anions in cells, and its staining results show that the superoxide anions in the husk at the top of the paa4 panicle are higher than those in the wild type at the 5cm stage, 7cm stage and 9cm stage ( Figure 4 H1 to J2). In addition, the inventors also measured the hydrogen peroxide and malondialdehyde contents in the husks at the top of the wild-type and paa4 ears. The results showed that the hydrogen peroxide content in the husks at the top of the paa4 ears was significantly higher than that in the wild-type ( Figure 4 K). The MDA content of the husk at the top of the paa4 panicle was only significantly different from that of the wild type at the 7 cm stage, but it also reached a very significant difference at the 9 cm stage ( Figure 4 The above results showed that reactive oxygen species began to accumulate at the 5-cm stage in the husk at the top of the paa4 panicle, and reached a peak at the 7-cm stage.

[0039] Then, the terminal deoxynucleotidyl transferase-mediated dUTP labeling (TUNEL) assay was used to detect the husks at the top of the spike of the wild type and paa4 at the 5 cm, 7 cm, and 9 cm stages. The results showed that no apoptotic signal was observed in the husks at the top of the spike of the wild type at the 5 cm, 7 cm, and 9 cm stages ( Figure 5 A, A1, D, D1, G, G1). A small amount of apoptosis signal appeared in the husk at the top of the paa4 panicle at the 5 cm stage ( Figure 5 B to C2), there are a lot of strong apoptosis signals in the apical husk of paa4 panicle at the 7 cm stage ( Figure 5 E to F2), the apoptosis signal in the husk at the top of the paa4 panicle at the 9 cm stage began to decrease, although it was not as strong as at the 7 cm stage, it still filled the entire husk ( Figure 5H to I2). The expression levels of programmed cell death and reactive oxygen species-related genes at the tips of wild-type and paa4 spikes were further analyzed. OsVPE2 and OsVPE3 have been reported to be associated with programmed cell death. OsVPE2 was upregulated at the tips of paa4 spikes at the 7 cm stage, and OsVPE3 was upregulated at the tips of paa4 spikes at the 7 cm and 9 cm stages ( Figure 5 J, K). In addition, OsCATA and OsCATB are involved in the removal of hydrogen peroxide. OsCATA and OsCATB are highly expressed at the apex of paa4 at the 7cm and 9cm stages ( Figure 5 L, M), indicating that there is a large amount of hydrogen peroxide at the tip of the paa4 ear that needs to be removed, which is consistent with the hydrogen peroxide content measured in the previous article. In summary, when the paa4 ear develops to the 5-7 cm stage, reactive oxygen stress appears at the tip of the ear, causing programmed cell death, and eventually leading to the cessation of husk development at the tip of the ear until it degenerates and dies.

[0040] (3) Positional cloning of PAA4

[0041] In order to accurately clone PAA4, its genetic regulation pattern must first be determined. The inventors prepared a population of Jinhui 10 (indica background) / paa4 and a hybrid combination. The F 1 All generations had normal phenotypes. 2 The segregation ratio of normal ear development and ear degeneration in the first generation was 3:1 (χ 2 =0.17<χ 2 0.05 =3.84), indicating that it is regulated by a pair of single recessive nuclear genes. 2 The plants with ear degeneration in the first generation all showed semi-dwarf traits, indicating that both plant height and ear degeneration phenotypes were caused by the PAA4 mutation. Then, the PAA4 gene was located and the F 2 260 panicle degenerate plants in the first generation were selected to locate the candidate gene. The candidate gene was located on chromosome 4 and linked to the LR-14 marker. RM6909, RM5506, RM6057, D4C-3 and D4C-4 were used to locate it within the 75kb interval at the end of the long arm. Finally, DNA sequencing was performed on this interval, and it was found that there was a base substitution in the gene LOC_Os04g56530 encoding MAPKKK in this interval ( Figure 6 A).

[0042] To verify the results of gene localization, the inventors sequenced the full length of LOC_Os04g56530. The results showed that there was a base substitution from G (SEQ ID No. 1) to A (SEQ ID No. 3) in the 19th exon of the gene ( Figure 6B) The inventors then transferred the normal function of PAA4 in the wild type into the mutant to form a complementary transgenic plant paa4 com . For wild type, paa4 and paa4 com Observation of the spike tip at maturity revealed that paa4 com There is no degeneration of the spike tip, which is the same as the wild type ( Figure 6 C to F). Comparison of wild type and paa4 com DNA sequencing results of LOC_Os04g56530 revealed paa4 com The mutation site in the middle is double peak, proving that paa4 com There is a normal function of PAA4 protein in Figure 6 G). The spike degeneration rate was statistically analyzed and it was found that WT and paa4 com There was no significant difference between Figure 6 H). The above results prove that LOC_Os04g56530 is the target gene of paa4, and its mutation will lead to degeneration of the ear apex.

[0043] Example 2: Study on the regulatory mechanism of PAA4

[0044] (1) Analysis of PAA4 protein structure

[0045] The PAA4 gene encodes OsMAPKKKε, a member of the MEKK family kinase. Sequence analysis determined that the N-terminal region (19-247a) is a typical kinase domain, and the ARM domain is located at 605-645aa and 1091-1176aa ( Figure 7 A). The paa4 mutation is located at amino acid position 791, resulting in a change from glycine (G) (SEQ ID No. 2) to aspartic acid (D) (SEQ ID No. 4). Phylogenetic analysis showed that PAA4 proteins clustered into two different evolutionary branches, indicating strong functional conservation in both monocots and dicots ( Figure 7 B). Protein sequences of the mutation site in monocots and dicots were then compared and it was found that the glycine at this site is conserved in all analyzed species ( Figure 7 C), which suggests that this mutation may seriously affect the function of PAA4. The mutation of paa4 occurs at the 791st amino acid of OsMAPKKKε, which is a currently unknown domain. The protein structure was predicted using Swiss-model, and the results showed that there was no significant change in the tertiary structure between PAA4 and its mutation ( Figure 7 C) For the mutation site, the amino acid in WT is Gly, while the amino acid in paa4 is Asp.

[0046] (2) PAA4 expression pattern

[0047] To explore the expression pattern of PAA4 in rice, RT-PCR was used to detect the expression of PAA4 in various rice tissues. RT-PCR results showed that PAA4 was expressed in various tissues, including roots, stems, leaves, sheaths, panicles, etc. ( Figure 8 A). The inventors then connected the PAA4 promoter to the GUS reporter gene, and the GUS staining results also supported the above results ( Figure 8 B to G). In addition, PAA4 was expressed in large amounts in the glume and in very small amounts in the glumes and anthers ( Figure 8 H, I).

[0048] The inventors added GFP to the full-length CDS of PAA4 to form a fusion protein PAA4::GFP, which was then transferred into rice protoplasts. 2 As a nuclear marker, the GFP signal of PAA4 was observed to be partially related to H 2 The overlap of group B proteins proves that they are distributed in the nucleus, and the remaining signals are distributed in the cytoplasm ( Figure 8 J to M).

[0049] (3) PAA4 participates in reactive oxygen species pathways

[0050] To investigate the role of PAA4 in the reactive oxygen species pathway, the inventors performed transcriptome sequencing analysis on the spike tips of wild-type and paa4. After filtering the raw data, a total of 2825 differentially expressed genes were detected in paa4, including 2014 upregulated genes and 811 downregulated genes. In addition, GO enrichment analysis found that these differentially expressed genes were associated with the cellular detoxification pathway (GO:1990748)( Fig. 9 A) and antioxidant activity (GO:0016209). Given the overlap between genes involved in antioxidant activity and genes in cellular detoxification pathways, the inventors' subsequent analysis will focus on cellular detoxification pathways. There were 23 differentially expressed genes in the intracellular detoxification pathway, among which LOC_Os08g41090, LOC_Os07g06190, and LOC_Os03g36560 were up-regulated, while LOC_Os01g53294 (OsrbohA), LOC_Os01g61880 (OsrbochE), LOC_Os08g35210 (OsrbophF), LOC_Os09g26660 (OsrbothG), LOC_Os01g15830 (OsPOX1), LOC_Os04g14680 (OsAPx3), and LOC_Os03g13210 (OsPRX38) were down-regulated. Figure 8B). It has been reported that respiratory burst oxidase homologs (Rbohs) are involved in the production of reactive oxygen species, and OsPOX1, OsAPx3, and OsPRX38 are peroxidases that eliminate hydrogen peroxide. In addition, genes such as peroxidase (LOC_Os02g09940, LOC_Os06g09610) and peroxidase precursors (LOC_Os10g02040, LOC_Os02g14160, LOC_Os01g36240, etc.) were upregulated ( Fig. 9 B) Subsequent RT-PCR validation of genes involved in detoxification and antioxidant activity confirmed their upregulation ( Fig. 9 CJ). These findings suggest that PAA4 is involved in the ROS pathway.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of the PAA4 gene in regulating rice panicle development, characterized in that: The amino acid sequence of the protein encoded by the PAA4 gene is shown in SEQ ID No.

2.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the PAA4 gene coding region is shown in SEQ ID No.

1.

3. The use according to any one of claims 1 and 2, characterized in that: The application is the application of the PAA4 gene in regulating rice length, panicle length, number of branches and stalks, and number of grains per panicle.

4. Application of the protein encoded by the PAA4 gene in regulating the development of rice panicles, characterized in that: The amino acid sequence of the protein encoded by the PAA4 gene is shown in SEQ ID No.

2.

5. The use according to claim 4, characterized in that: The application is the application of the protein encoded by the PAA4 gene in regulating the length, panicle length, number of branches and stalks, and number of grains per panicle of rice.

6. Application of the PAA4 gene in rice panicle trait selection, characterized in that the amino acid sequence of the protein encoded by the PAA4 gene is shown in SEQ ID No.

2.

7. The use according to claim 6, characterized in that: The nucleotide sequence of the PAA4 gene coding region is shown in SEQ ID No.

1.

8. Application of the protein encoded by the PAA4 gene in rice panicle trait breeding, characterized in that: The amino acid sequence of the protein encoded by the PAA4 gene is shown in SEQ ID No.2.

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