CN111424080A

CN111424080A - Application of substance for detecting HSF4 gene mutation in diagnosis of giant panda cataract

Info

Publication number: CN111424080A
Application number: CN202010263966.XA
Authority: CN
Inventors: 由玉岩; 柏超; 刘学锋; 王伟; 贾婷; 张成林; 李晓光; 尹彦强; 夏茂华; 卢岩; 普天春; 陈玉村; 王利勤; 修云芳; 杜洋; 马涛; 刘彦晖
Original assignee: Beijing Zoo
Current assignee: Beijing Zoo
Priority date: 2020-04-07
Filing date: 2020-04-07
Publication date: 2020-07-17
Anticipated expiration: 2040-04-07
Also published as: CN111424080B

Abstract

The invention discloses application of a substance for detecting HSF4 gene mutation in diagnosis of giant panda cataract. The invention discloses a gene mutation related to giant panda cataract, namely mutation of nucleotide 1132 th position of HSF4 gene from C to T, which causes mutation of amino acid residue 378 of HSF4 protein coded by the gene from Arg to Trp, wherein the mutation exists in a giant panda new star with cataract, and the mutation does not exist in the rest of giant pandas, thereby indicating that the mutation can be used for diagnosing the giant panda cataract or evaluating the risk of the giant panda cataract.

Description

Application of substance for detecting HSF4 gene mutation in diagnosis of giant panda cataract

Technical Field

The invention relates to the field of animal medicine, in particular to application of a substance for detecting HSF4 gene mutation in diagnosis of giant panda cataract.

Background

Most cataracts are associated with aging, environmental factors or genetic factors. Approximately one third of cataracts have a significant genetic component, some due to disruption of normal lens development, some due to mutations in the lens proteins themselves or other proteins required for normal lens physiology, including blockade of oxidative stress pathways. The cumulative response of oxidative damage can also gradually damage the lens, or lead to accumulation of pigment or aggregation of lens proteins, which are the main components for maintaining lens transparency and refraction. However, genetic mutations affect lens proteins, as well as mutations affecting structural proteins of the connexin and myelin precursor families, gap connexins, or stress response components. Some mutations affect transcription factors expressed in the lens, such as FOXE3, HSF4, MAF and PITX3, because these mutations may perturb the activity of many downstream genes.

Cataracts are a common cause of vision impairment and blindness in humans, other primates and companion animals, and most cataract studies have therefore focused on these species or murine disease models. However, containment of large wild animals tends to be longer than field congeners, and cataract development is also prominent with age. More than 20% of the elderly pandas (Ailuropoda melanoleuca) in china suffer from cataracts, which have a significant impact on their quality of life. Panda cataract prevalence increases with age of animals, which severely affects their health and well-being. However, few studies have focused on the cause and treatment of cataracts in pandas. Due to the special shape of the giant panda lens, the curvature of the lens is difficult to measure in vitro, and the lens cannot be manufactured and operated. Therefore, the causes of cataract, prevention strategies and treatment methods of captive pandas are awaiting further research.

The heat shock transcription factor 4(HSF4) is a pathogenic gene closely related to cataract. HSF4 differs from other HSFs (including HSF1, HSF2, HSF3) in that it lacks the C-terminal HR-C domain. HSF4 can respond to various stress stimuli and protect cells from proteotoxic damage, thereby participating in the regulation of differentiation and development. Many previous studies reported that HSF4 is expressed primarily in the lens of the eye and plays an important role in lens fiber cell differentiation and early cataract formation). Mutations in HSF4 in the DNA binding domain may lead to autosomal dominant cataracts, while mutations in HSF4 in other domains may lead to cryptic cataracts. In addition, the HSF4 mutation promoted lens enucleation and resulted in lens protein loss and cataract development. The HSF4 mutation was also found to be associated with age-related cataracts.

Disclosure of Invention

The invention aims to solve the technical problem of how to diagnose the cataracts of pandas.

In order to solve the technical problem, the invention firstly provides any one of the following applications of a substance for detecting HSF4 gene mutation:

x1, preparing a product for diagnosing or assisting in diagnosing cataracts of pandas;

x2, preparing a product for evaluating or assisting in evaluating the risk of developing cataract of the pandas to be tested;

x3, preparing a product for evaluating or assisting in evaluating the risk of developing cataract of the panda offspring to be tested;

x4, preparing a product for diagnosing or assisting in diagnosing giant panda lens-soluble glaucoma;

x5, preparing a product for evaluating or assisting in evaluating the risk of the pandas to be tested for suffering from the lens-soluble glaucoma;

x6, preparing a product for evaluating or assisting in evaluating the risk of the offspring of pandas to be tested suffering from lens-soluble glaucoma;

the HSF4 gene is mutated into the nucleotide of 1132 th site of the HSF4 gene from C to T (namely the nucleotide of 31 st site of a sequence 1 in a sequence table is mutated from C to T).

In the above application, the substance for detecting HSF4 gene mutation comprises a primer pair capable of amplifying a DNA fragment containing the HSF4 gene mutation.

In the application, the primer pair can be composed of two single-stranded DNAs shown as

sequences

3 and 4 in a sequence table.

The substance for detecting the mutation of the HSF4 gene can be composed of the primer pair and other reagents required for PCR amplification besides the primer.

The invention also provides any one of the following applications of the substance for detecting the mutation of the HSF4 protein:

the HSF4 protein is mutated into the HSF4 protein, the 378 th amino acid residue is mutated into Trp from Arg (namely, the 11 th amino acid residue in the sequence 2 in the sequence table is mutated into tryptophan residue from arginine residue).

The substance for detecting mutation of the HSF4 protein may be a substance capable of specifically recognizing mutation of the HSF4 protein or the HSF4 protein.

The product may be a kit as hereinbefore described.

The invention also provides a kit, which comprises the substance for detecting the mutation of the HSF4 gene or the substance for detecting the mutation of the HSF4 protein.

The kit can have any one of the following uses:

y1, diagnosis or auxiliary diagnosis of cataracts of pandas;

y2, evaluating the risk of the pandas to be detected suffering from cataract;

y3, evaluating the risk of the offspring of the pandas to be detected to suffer from cataract;

y4, diagnosis or aid diagnosis of panda lens-dissolving glaucoma;

y5, evaluating the risk of the pandas to be detected to suffer from lens-dissolving glaucoma;

y6, evaluating the risk of the panda offspring suffering from lens-dissolving glaucoma to be tested.

The kit can also comprise a carrier which is recorded with a1) or a2) or a3) as follows:

a1) the following a11) or a 12):

a11) if the genome of the panda to be tested contains the HSF4 gene mutation, the panda to be tested has or is candidate to have cataract;

a12) if the genome of the panda to be detected does not contain the HSF4 gene mutation, the panda to be detected does not suffer from cataract or candidate does not suffer from cataract;

a2) the following a21) or a 22):

a21) if the genome of the panda to be tested contains the HSF4 gene mutation, the risk of the panda to be tested suffering from cataract is higher than that of the normal panda;

a22) if the genome of the panda to be detected does not contain the HSF4 gene mutation, the risk of the panda to be detected suffering from cataract is not higher than that of the normal panda;

a3) the following a31) or a 32):

a31) if the genome of the panda to be tested contains the HSF4 gene mutation, the risk of cataract of the offspring of the panda to be tested is higher than that of the normal panda;

a32) if the genome of the panda to be tested does not contain the HSF4 gene mutation, the risk of cataract of the offspring of the panda to be tested is not higher than that of the normal panda.

The kit may further include a vector carrying b1) or b2) or b3) as follows:

b1) the following b11) or b 12):

b11) if the genome of the panda to be tested contains the HSF4 protein mutation, the panda to be tested has or is candidate to have cataract;

b12) if the genome of the panda to be tested does not contain the HSF4 protein mutation, the panda to be tested does not suffer from cataract or candidate does not suffer from cataract;

b2) the following b21) or b 22):

b21) if the genome of the panda to be tested contains the HSF4 protein mutation, the risk of the panda to be tested suffering from cataract is higher than that of the panda to be tested;

b22) if the genome of the panda to be detected does not contain the HSF4 protein mutation, the risk of the panda to be detected suffering from cataract is not higher than that of the normal panda;

b3) the following b31) or b 32):

b31) if the genome of the panda to be tested contains the HSF4 protein mutation, the risk of cataract of the offspring of the panda to be tested is higher than that of the normal panda;

b32) if the genome of the panda to be tested does not contain the HSF4 protein mutation, the risk of the offspring of the panda to be tested suffering from cataract is not higher than that of the normal panda.

The kit may further comprise a vector carrying c1) or c2) or c3) as follows:

c1) the following c11) or c 12):

c11) if the genome of the panda to be detected contains the HSF4 gene mutation, the panda to be detected has or is candidate to have the lens dissolving glaucoma;

c12) if the genome of the panda to be detected does not contain the HSF4 gene mutation, the panda to be detected does not suffer from or candidate does not suffer from lens-dissolving glaucoma;

c2) the following c21) or c 22):

c21) if the genome of the panda to be detected contains the HSF4 gene mutation, the risk of the panda to be detected suffering from the lens-soluble glaucoma is higher than that of the panda to be detected suffering from the normal fel;

c22) if the genome of the panda to be detected does not contain the HSF4 gene mutation, the risk of the panda to be detected suffering from the lens-soluble glaucoma is not higher than that of the panda to be detected suffering from the normal fel;

c3) the following c31) or c 32):

c31) if the genome of the panda to be detected contains the HSF4 gene mutation, the risk that the offspring of the panda to be detected has the lens-soluble glaucoma is higher than that of the normal panda;

c32) if the genome of the panda to be detected does not contain the HSF4 gene mutation, the risk that the offspring of the panda to be detected has the lens-soluble glaucoma is not higher than that of the normal panda.

The kit may further comprise a vector carrying d1) or d2) or d3) as follows:

d1) the following d11) or d 12):

d11) if the genome of a panda to be tested contains the HSF4 protein mutation, the panda to be tested has or is candidate to have the lens-dissolving glaucoma;

d12) if the genome of the panda to be tested does not contain the HSF4 protein mutation, the panda to be tested does not suffer from or is not candidate to suffer from lens-soluble glaucoma;

d2) the following d21) or d 22):

d21) if the genome of the panda to be detected contains the HSF4 protein mutation, the risk of the panda to be detected suffering from the lens-soluble glaucoma is higher than that of the panda to be detected suffering from the normal fel;

d22) if the genome of the panda to be detected does not contain the HSF4 protein mutation, the risk of the panda to be detected suffering from the lens-soluble glaucoma is not higher than that of the panda to be detected suffering from the normal fel;

d3) the following d31) or d 32):

d31) if the genome of the panda to be tested contains the HSF4 protein mutation, the risk that the offspring of the panda to be tested has the lens-soluble glaucoma is higher than that of the normal panda;

d32) if the genome of the panda to be detected does not contain the HSF4 protein mutation, the risk that the offspring of the panda to be detected has the lens-soluble glaucoma is not higher than that of the normal panda.

The kit can be composed of the substance for detecting the mutation of the HSF4 gene or the substance for detecting the mutation of the HSF4 protein, and also can be composed of the substance for detecting the mutation of the HSF4 gene or the substance for detecting the mutation of the HSF4 protein and the carrier which is recorded with a1) or a2) or a3) or b1) or b2) or b3) or c1) or c2) or c3) or d1) or d2) or d 3).

In the present invention, the cataract may be a cortical cataract. The cataract may be a high maturity stage cortical cataract. The cataract may be age-related cataract induced by gene mutation.

The inventor of the invention screens a gene mutation related to giant panda cataract and lens-soluble glaucoma by a method combining a functional candidate gene screening method and bioinformatics analysis, namely, the 1132 th nucleotide of the HSF4 gene is mutated from C to T, so that the 378 th amino acid residue of the encoded HSF4 protein is mutated from Arg to Trp, the mutation exists in giant panda neosatellite with cataract and lens-soluble glaucoma, and the mutation does not exist in the rest giant pandas, thereby showing that the mutation can be used for diagnosing or evaluating the risk of developing the giant panda cataract and the lens-soluble glaucoma.

Drawings

Fig. 1 left eye of female panda Xinxing.

FIG. 2 location and identification of mutations in the New Star HSF4 gene. (a) The structure of giant panda HSF4 gene and the corresponding protein. Exons 10-12 (the region where PCR primers were used to detect mutations) were amplified, exons are shown as open boxes, introns are shown as lines, arrows to the right and left indicate the upstream and downstream primers, respectively, of primer pair HSF4-10-12, down arrows indicate the position of the mutation, and a line at 329 indicates a glycine-rich fragment unique to panda HSF 4. (b) Sequence tracing is performed in the 17bp region of the mutation site, 5 unaffected individuals (upper) of the site are compared with new star individuals (lower), and heterozygote c.1132c > T mutations are shown by arrows.

Figure 3 effect of substitution of highly conserved arginine residues with tryptophan between HSF4 isoform specificity and the downstream hydrophobic region. (a) Multiple alignments of the highly conserved sequence of 25 amino acid residues in four homologous genes of HSF4 (mouse, dog, human and xinxing) showed that panda R378W substitutions affected the conserved arginine residue (corresponding to human residue R371) in all species. (b) The increase in overall hydrophobicity was confirmed by a primary scale analysis of the human protein with an allelic mutation (R371W) (boxed).

FIG. 4 is based on the structure prediction of giant panda HSF4 protein missense mutation R378W. (a) The alteration of the surface structure is visualized using the Discovery Studio visualization tool. (b) The interaction between amino acid side chains was predicted using the THREADER and modeler.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

Example 1 discovery of Gene mutations associated with cataracts in pandas and Leptoglaucoma

Materials (I) and (II)

Giant panda with cataract: xinxing, born in 1982, from Chongqing zoo, female, father and mother from field, since 2010, cataract developed, both sides of cornea shriveled basically, poor eyesight, slow movement and dull expression. In addition, 5 pandas were selected as controls from Chongqing, Chengdu and Beijing zoos.

The information for each panda is as follows:

panda name	Year of birth	Origin of origin	Disease states	Sex
					Girl and old jacket	1999 year	Chongqing zoo	Healthy panda without cataract	Female
Freezing point	Year 2000	CHENGDU ZOO	Health without cataract	Male sex
					Ya A	1990 s	CHENGDU ZOO	Health without cataract	Female
Happy music	In 1986	BEIJING ZOO	Cataract patients	Female
					Jane	1993	BEIJING ZOO	Cataract patients	Female
New star	In 1982	Chongqing zoo	Cataract patients	Female

Wherein, happy: in 2011, two types of cataract, namely left-eye cortical cataract and right-eye nuclear cataract, rarely occur simultaneously.

And (4) Jane: birth in 1993, onset of cataract in 2015, as shown in fig. 1. Unilateral eye disease, characterized by the early stage of cortical cataract, with lenticular opacity around the cortex, feathery opacity. Vision deteriorates.

The samples used hereinafter were all those collected from the panda normal physical examination.

Second, detection method

According to the previous report, 11 highly homologous cataract candidate genes of animals are selected for mutation screening, and the 11 genes are CRYAB gene, CRYBA1 gene, CRYBB1 gene, CRYGC gene, HSPB6 gene, HSPB7 gene, HSPB9 gene, GJA3 gene, AQP3 gene, MIP gene and HSF4 gene. The genomic DNA of each panda was amplified by PCR using the primers for these genes shown in Table 1.

The PCR reaction systems (total volume 25. mu.l) were 2 × PCR mixtures (containing Taq DNA polymerase, dNTPs, MgCl)₂Reaction buffer, enhancer and optimizer for PCR reaction and stabilizer) (Tiangen Biochemical technology (Beijing) Ltd., product No. KT201), 1. mu.l each of forward primer and reverse primer, 2. mu.l (20ng) of genomic DNA, and the balance of water. And (3) PCR reaction conditions: denaturation at 95 ℃ for 5 min; then 34 cycles of annealing at 95 ℃ for 30s at the corresponding annealing temperature for 30s and annealing at 72 ℃ for 30s are carried out; finally, extension was carried out at 72 ℃ for 10 minutes.

The resulting PCR products were sequenced using an ABI 3730 automated sequencer (PE Biosystems, Foster City, Calif.). Sequencing results were analyzed using Chromas 2.33 and compared to reference sequences in NCBI databases.

TABLE 1 PCR primers for screening candidate Gene mutations

Third, bioinformatics analysis

Multiple sequence alignments of the amino acid sequences in HSF4 from several different species were analyzed by C L C Free Workbench 4.5.1 software (C L C bio, Aarhus, denmark).

The THREADER 3.5(1-3) and Modeller 9.22(4) predicted structural and functional amino acid substitutions for the HSF4 mutation. The Discovery Studio Visualizer showed hydrophobic interactions and structural changes between mutant HSF4 and wild type.

Fourthly, the result

1. The clinical findings are as follows: new star patients were examined at 28 years of age and were first diagnosed with cataracts, with the left eye shown in FIG. 1. It is in the high maturation stage of cortical cataracts, characterized by a narrowing of the lens capsule, a deepening of the anterior chamber, a subsidence of the lens nucleus, a deposition of lens cortical particles in the anterior chamber angle, a blockage of the trabecular meshwork and secondary glaucoma (clinically known as phaco-lytic glaucoma). Clouding of the lens, dislocation into the anterior chamber, leukoplakia. The hardness of the lens nucleus is II.

2. Mutation analysis: a new pathogenic mutation is identified by detecting the sequences of 11 candidate genes of each panda (figure 2), wherein c.1132C > T missense mutation in exon 10 of the HSF4 gene is that the 1132 th nucleotide in the 10 th exon of the HSF4 gene is mutated from C to T (namely the 31 st nucleotide in the sequence 1 in the sequence table is mutated from C to T, and the sequence 1 is a new star sequence), so that the 378 position of arginine residue in the 378 position of the encoded HSF4 protein is mutated into tryptophan residue (378R > W) (namely the 11 th nucleotide in the sequence 2 in the sequence table is mutated from arginine residue to tryptophan residue, and the sequence 2 is a new star sequence). The mutation is located in the PCR amplified product of HSF4-10-12, which is a primer pair consisting of sequence 3 and sequence 4 in Table 1. The mutation was present only in pandas named "new star" and was heterozygous, and was not detected in the other 5 individuals.

3. Bioinformatics analysis:

the inventors selected HSF4 genes from other mammals for conservation analysis, and the results showed that the sequences of three mammals present highly conserved regions in many species, arginine at position 378 of HSF4 is very conserved (fig. 3, C L CMain Workbench software).

In addition, the Modeller 9.22(4) was used to predict the structural and functional effects of the HSF4 mutein. The template structure was searched using the streamer 3.5 (fig. 4). Streptococcus pneumoniae hyaluronidase (PDB code 1N7O) (5) was selected as a modeling template. As shown in fig. 4 (b), Arg378 in wild-type HSF4 was changed to Trp378, resulting in a change in hydrophilicity. The change in interaction indicates that Arg378 forms two hydrogen bonds with Gly372 and Pro398 to form a cation-pi interaction with normal Pro 398. However, the Phe388 mutation in HSF4 performed two pi-pi stacking interactions with Pro 412. These predictions suggest that this variant may have an effect on protein structure or stability, leading to the achievement of HSF4 function in the corresponding abnormality, leading to the development of cataracts.

HSF4 is an essential factor for normal lens development and fibroblast differentiation (fujimoto et al, 2004; min et al, 2004; gao et al, 2017), is associated with several autosomal dominant inheritance of human cataracts (marner, 1949; bu et al, 2002), but is also associated with age-related cataracts (bu et al, 2002; ke et al, 2006; enoki et al, 2010; lv et al, 2014). many cataracts are associated with abnormalities of crystallin genes. in HSF4 gene knockout experiments, HSF4 knockout mice with different genetic backgrounds, different types of crystallins are expressed at different levels down-regulated, including gamma (AF) crystallin genes, α B-crystallin genes and 20154A-crystallin members (fujimoto et al, 2002) and HSF α A-crystallin genes (fujimoto et al, 2002. HSF4 can also regulate the expression of fibrocyte and downstream regulators thereof by activating p53, and the early regulator thereof, thus the early cataracts may cause the development of cataract in HSF 493, the cataract, the early cataract, the cataract may be caused by combining with the early cataracts of the age-related cataracts (bulla cataract, the cataract expression of the HSF 493, the cataract caused by the early cataract observed by the activation of the early cataract, the mutation of the early cataract, the cataract caused by the mutation of the early cataract, the early cataract caused by the mutation of the early cataract, the early cataract caused by the cataract, the early cataract, the mutation of the early cataract caused by the mutation of the early cataract.

Mutations at different positions of HSF4 have different clinical effects, and a recent literature survey summarized 16 studies reporting 14 unique pathogenic mutations in the human HSF4 gene, most of which are missense mutations, as well as individual frameshift and nonsense mutations (anand et al, 2018). Cataracts also occur as a result of homozygous splicing mutations inherited from autosomal recessive genes (smaoui et al, 2004). Most mutations occur in the DNA binding domain of HSF4 (represented by exons 3-6), and a few mutations occur in the oligomerization domain required to form trimers (exons 6-8) or downstream hydrophobic repeats (exons 12-14). At present, the mutation position of HSF4b has not been reported, and pathogenic mutation has not been found in conserved amino acids between isomer specific region and the downstream hydrophobic region repeat. Other mutations have been reported in the literature, but these are also located mainly in the DNA binding domain (cao et al, 2018; berry et al, 2018; li et al, 2019) and in a region very close to the C-terminus of the protein (zhang et al, 2018). Therefore, the discovery of pathogenic mutations between the specific region of panda HSF4 isomer and the downstream hydrophobic region indicates that a new pathogenesis of the disease may exist.

The present inventors found that the heterozygote 1132C > T mutation resulted in a R378W substitution, replacing the arginine residue that is completely conserved among all mammalian orthologies, this residue is also located in the highly conserved region of the isoform HSF4b, HSF4b being the major splice variant expressed in the lens, located within the highly conserved segment before the downstream hydrophobic region, thus, the major region as a lens-specific subtype (with important lens-specific functions) has not been associated with any form of human cataract a number of studies have described pathogenic mutations in other parts of the human protein caused by arginine residue substitutions, but no corresponding human mutations (R371W) have been reported so far, including R73 ke H (ke et al, 2006; shi et al, 2008), R73L (119 o et al, 2018), R110C (liu et al, 2015), R119R C (bu et al, 2002; hansen et al, 2009, 119R 9 (et al), 119 et al, 2019 (binding to all of these mutations that affect the structure of the target DNA binding to the target DNA 175, which has been reported to all of the oligo R371R 175, wo 103, 175.

Equivalent human mutations analyzed in bioinformatics (R371W) show that arginine replacement by increased hydrophobicity, even though its DNA binding capacity and trimer forming capacity are not affected, may affect the binding of HSF4b to its interaction partner one potential reason for no similar mutation in humans is that panda R W mutation is just downstream of the glycine-rich insert (GGGAPRG), which is known to be unique to pandas even absent in close species such as grey bear, the presence of multiple glycines and one proline residue may have profound effects on folding of the polypeptide backbone, compared to homologous genes lacking this sequence, the effect of nearby mutations converting arginine to tryptophan may therefore be affected by the background of the glycine-rich sequence — F4 is known to be affected by BC L (e.a., 2005), dual phosphatase 26 and mitogen-activated protein kinase (MAPK) family members (R and michi), brchia 2006, brkol, cd 8, H8, cd 94, cd 8, cd.

In summary, the inventors have discovered a new mutation in exon 10 of the neonatal panda HSF4 gene that is not found in the other 5 pandas, nor has such a mutation been reported in humans or other mammals, where most disease alleles map to DNA binding domains, or more rarely, oligomeric domains or downstream hydrophobic repeats. The R378W substitution was mapped to a highly conserved fragment between the isoform-specific region found in HSF4b and the downstream hydrophobic region. This mutation is unlikely to affect DNA binding or trimer formation, and therefore it would affect the interaction between HSF4 and its binding protein. The discovery of the invention reveals a new genetic determinant which is helpful for predicting the risk of suffering cataract of pandas or assisting in diagnosing the cataract of pandas.

<110> Beijing zoo

<120> application of substance for detecting HSF4 gene mutation in diagnosis of giant panda cataract

<160>4

<170>PatentIn version 3.5

<210>1

<211>125

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<213> panda (Ailuropoda melanoleuca)

<400>1

aggggaactc tgggcctgga gaggggggga tggagccccg agcatctgct acctcccatg 60

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Arg Gly Thr Leu Gly Leu Glu Arg Gly Gly Trp Ser Pro Glu His Leu

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

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Gly Pro Leu Asp Val Ser Thr Pro

35 40

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atcccaagac cccagttcca t 21

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Claims

1. The following application of the substance for detecting HSF4 gene mutation is as follows:

the HSF4 gene is mutated into the 1132 th nucleotide of the HSF4 gene, and the nucleotide is mutated from C to T.

2. Use according to claim 1, characterized in that: the substance for detecting the mutation of the HSF4 gene comprises a primer pair capable of amplifying a DNA fragment containing the mutation of the HSF4 gene.

3. Use according to claim 2, characterized in that: the primer pair consists of two single-stranded DNAs shown as sequences 3 and 4 in a sequence table.

4. The following application of the substance for detecting the mutation of the HSF4 protein comprises the following steps:

the HSF4 protein is mutated into the HSF4 protein, wherein the 378 th amino acid residue of the HSF4 protein is mutated from Arg to Trp.

5. A kit comprising the substance for detecting mutation of HSF4 gene according to any one of claims 1-3 or the substance for detecting mutation of HSF4 protein according to claim 4.

6. The kit of claim 5, wherein: the kit has any one of the following uses:

y1, diagnosis or auxiliary diagnosis of cataracts of pandas;

y2, evaluating the risk of the pandas to be detected suffering from cataract;

y4, diagnosis or aid diagnosis of panda lens-dissolving glaucoma;

7. The kit according to claim 5 or 6, characterized in that: the kit also comprises a carrier which is recorded with a1) or a2) or a3) as follows:

a1) the following a11) or a 12):

a11) the panda genome to be tested containing the HSF4 gene mutation of claim 1, wherein the panda to be tested has or is candidate for having cataract;

a12) if the genome of the panda to be tested does not contain the HSF4 gene mutation in the claim 1, the panda to be tested does not suffer from cataract or does not candidate cataract;

a2) the following a21) or a 22):

a21) the genome of a panda to be tested contains the HSF4 gene mutation in the claim 1, and the risk of the panda to be tested suffering from cataract is higher than that of a normal panda;

a22) if the genome of the panda to be tested does not contain the HSF4 gene mutation in the claim 1, the risk of the panda to be tested suffering from cataract is not higher than that of the normal panda;

a3) the following a31) or a 32):

a31) the genome of a panda to be tested contains the HSF4 gene mutation in the claim 1, and the risk of cataract of the offspring of the panda to be tested is higher than that of the offspring of the normal panda;

a32) if the genome of the panda to be tested does not contain the HSF4 gene mutation in the claim 1, the risk of the offspring of the panda to be tested suffering from cataract is not higher than that of the normal panda.

8. The kit according to claim 5 or 6, characterized in that: the kit also comprises a carrier recorded with b1) or b2) or b3) as follows:

b1) the following b11) or b 12):

b11) the panda genome to be tested comprising the HSF4 protein mutation of claim 4, wherein said panda to be tested has or is a candidate for developing cataract;

b12) the panda genome to be tested does not contain the HSF4 protein mutation of claim 4, wherein the panda to be tested does not suffer from or is not candidate to suffer from cataract;

b2) the following b21) or b 22):

b21) the genome of a panda to be tested contains the HSF4 protein mutation described in claim 4, and the risk of suffering cataract of the panda to be tested is higher than that of a normal panda;

b22) the genome of a panda to be tested does not contain the HSF4 protein mutation of claim 4, and the risk of the panda to be tested suffering from cataract is not higher than that of a normal panda;

b3) the following b31) or b 32):

b31) the genome of a panda to be tested contains the HSF4 protein mutation of claim 4, and the risk of cataract of the offspring of the panda to be tested is higher than that of the offspring of the normal panda;

b32) if the genome of the panda to be tested does not contain the HSF4 protein mutation described in claim 4, the risk of the offspring of the panda to be tested suffering from cataract is not higher than that of the normal panda.

9. The kit according to claim 5 or 6, characterized in that: the kit also comprises a carrier recorded with c1) or c2) or c3) as follows:

c1) the following c11) or c 12):

c11) the panda genome to be tested comprising the HSF4 gene mutation of claim 1, said panda to be tested having or being candidate for having lens-dissolving glaucoma;

c12) the panda test does not have the HSF4 gene mutation of claim 1 in its genome, and said panda test does not have or is not a candidate for developing phacoellisolving glaucoma;

c2) the following c21) or c 22):

c21) the genome of a panda to be tested contains the HSF4 gene mutation in the claim 1, and the panda to be tested has higher risk of suffering from lens-dissolving glaucoma than a normal panda;

c22) the genome of a panda to be tested does not contain the HSF4 gene mutation in the claim 1, and the panda to be tested has the risk of having the lens-dissolving glaucoma which is not higher than that of a normal panda;

c3) the following c31) or c 32):

c31) the genome of a panda to be tested contains the HSF4 gene mutation in the claim 1, and the offspring of the panda to be tested has higher risk of having the lens-dissolving glaucoma than that of a normal panda;

c32) if the genome of the panda to be tested does not contain the HSF4 gene mutation in the claim 1, the offspring of the panda to be tested has the risk of the lens-dissolving glaucoma which is not higher than that of the normal panda.

10. The kit according to claim 5 or 6, characterized in that: the kit also comprises a carrier recorded with d1) or d2) or d3) as follows:

d1) the following d11) or d 12):

d11) the panda test having or being candidate for having lens-dissolving glaucoma containing in its genome the HSF4 protein mutation of claim 4;

d12) the panda test not suffering from or being a candidate for not suffering from lens-soluble glaucoma, wherein the genome of said panda test does not contain the HSF4 protein mutation of claim 4;

d2) the following d21) or d 22):

d21) the genome of a panda to be tested, which contains the HSF4 protein mutation of claim 4, has higher risk of developing the lens-soluble glaucoma than that of a normal panda;

d22) the genome of a panda to be tested does not contain the HSF4 protein mutation of claim 4, and the panda to be tested has the risk of having the lens-dissolving glaucoma which is not higher than that of a normal panda;

d3) the following d31) or d 32):

d31) the genome of a panda to be tested contains the HSF4 protein mutation of claim 4, and the offspring of the panda to be tested has higher risk of having the lens-dissolving glaucoma than that of a normal panda;

d32) the genome of a panda to be tested does not contain the HSF4 protein mutation of claim 4, and the offspring of the panda to be tested has the higher risk of having the lens-dissolving glaucoma than that of a normal panda.