Application of Aux / IAA gene of sisal hemp and protein of Aux / IAA gene in plants

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to a sisal hemp Aux / IAA gene and application of protein of the sisal hemp Aux / IAA gene in plants. The sisal hemp Aux / IAA gene provided by the invention comprises a first SNP (Single Nucleotide Polymorphism) site or a second SNP site. Research finds that the Aux / IAA gene can increase the number of the vascular tissues of the plant, construct a plant model related to the vascular tissues and cultivate the multi-vascular tissue plant; the first SNP site or the second SNP site on the gene is significantly related to the number of vascular bundles of sisal hemp leaves, and can be used for screening and cultivating multi-vascular tissue plants, thereby providing a theoretical basis and gene resources for genetic breeding.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology, and specifically relates to sisal hemp Aux / IAA Application of genes and their proteins in plants. Background Art

[0002] Aux / IAA The auxin / indole-3-acetic acid gene family is one of the key gene families in the auxin signaling pathway. The proteins encoded by them participate in the regulation of the auxin signaling pathway by specifically binding to ARF (Auxin response factor). Aux / IAA Whole genome analysis of gene families. The number of published plant genomes has increased year by year, making whole genome level research possible. Aux / IAA The evolutionary mechanism of gene families becomes possible. With the increase of biological information every year, Aux / IAA It is increasingly important to conduct in-depth molecular biological research on gene families and to analyze their mechanisms of regulating plant development.

[0003] Arabidopsis thaliana Aux / IAA The identification of gene functions started early, and the biological functions of many genes have been analyzed, such as regulating the tropism of apical buds and roots, elongation of hypocotyls, leaf shape development, and lateral root development. However, most of the related research in Arabidopsis involves auxin-mediated developmental regulation, and there are relatively few studies on specific agronomic traits. In recent years, with the in-depth development of crop molecular biology research, Aux / IAA Reports on gene regulation of agronomic traits are increasing. For example, OsIAA6 It can improve the drought tolerance of rice and regulate the number of tillers, corn IAA11 Participates in the regulation of vascular bundle development, BnaA3.IAA7 It can improve rapeseed plant type and increase yield, corn and peach Aux / IAA Genes involved in regulating pericarp development, etc. In addition, research reports on eucalyptus and poplar have confirmed PtoIAA9 and EgrIAA4 It plays an important regulatory role in the development of xylem.

[0004] Sisal ( Agave sisalana Perr. ex Engelm. ) is a hard leaf fiber crop that is widely grown in tropical and subtropical regions. It has a wide range of uses, mainly involving fisheries, navigation, aerospace, industry and mining, transportation, oil fields, textiles and other industries. Sisal is a unique leaf fiber crop among hemp crops, different from seed fiber crops represented by cotton and bast fiber crops represented by ramie and flax. Sisal fiber is mainly developed from leaf vascular tissue, and its fiber trait formation mechanism is not yet clear. Therefore, it is crucial to explore the functional genes that regulate vascular tissue traits for the genetic improvement of sisal fiber traits. There is currently no sisal Aux / IAAResearch on gene regulation of vascular tissue traits and molecular markers. Summary of the Invention

[0005] The object of the present invention is to provide sisal Aux / IAA The application of the gene and its protein in plants can be used to increase the amount of plant vascular tissue, construct plant models related to vascular tissue, and cultivate and screen plants with multiple vascular tissues.

[0006] The present invention provides sisal Aux / IAA Gene, the sisal Aux / IAA The gene includes a first SNP site or a second SNP site; The sisal Aux / IAA The nucleotide sequence of the gene includes any one of (a1) to (a3): (a1). As shown in SEQ ID NO.1; (a2). The nucleotide sequence containing the first SNP site is shown in SEQ ID NO. 7; (a3). The nucleotide sequence containing the second SNP site is shown in SEQ ID NO.8.

[0007] Preferably, in the same plant germplasm, the number of vascular bundles in the leaves of the A genotype is significantly greater than the number of vascular bundles in the leaves of the G genotype; The A genotype is: sisal Aux / IAA The nucleotide sequence of the gene is shown in SEQ ID NO.7 or SEQ ID NO.8; The G genotype is: sisal Aux / IAA The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] The present invention provides the sisal hemp described in the above technical solution Aux / IAA The sisal Aux / IAA protein encoded by the gene has an amino acid sequence shown in SEQ ID NO.2.

[0009] The present invention provides a biomaterial, which comprises: Aux / IAA Recombinant expression vector or recombinant microorganism of the gene.

[0010] The present invention provides the sisal hemp described in the above technical solution Aux / IAA Application of the gene, the sisal Aux / IAA protein or the biological material in plants.

[0011] Preferably, the plant comprises one or more of sisal, corn, rice, tobacco and Arabidopsis.

[0012] Preferably, the application includes one or more of: increasing the amount of plant vascular tissue, constructing a plant model related to vascular tissue, and cultivating and screening plants with multiple vascular tissues.

[0013] Preferably, the plant vascular tissue includes: vascular bundles in leaves and / or vascular columns in rachis.

[0014] The present invention provides a method for cultivating multivascular tissue plants, comprising the following steps: Increase the sisal content in plants Aux / IAA The expression level of the gene or the content of the sisal Aux / IAA protein in the plant is increased to obtain a multivascular tissue plant.

[0015] The present invention provides a method for screening multidimensional tubular sisal, comprising the following steps: Using the DNA of the sisal to be tested as a template, PCR amplification and sequencing analysis were performed in sequence; When the nucleotide sequence of the PCR product is as shown in SEQ ID NO.7 or SEQ ID NO.8, the sisal to be tested is multivascular sisal; When the nucleotide sequence of the PCR product is as shown in SEQ ID NO. 1, the plant to be tested is oligovascular sisal.

[0016] Beneficial effects: The present invention provides sisal Aux / IAA Gene, the sisal Aux / IAA The gene includes a first SNP site or a second SNP site; the sisal Aux / IAA The nucleotide sequence of the gene includes any one of (a1)-(a3): (a1). As shown in SEQ ID NO.1; (a2). The nucleotide sequence containing the first SNP site is shown in SEQ ID NO.7; (a3). The nucleotide sequence containing the second SNP site is shown in SEQ ID NO.8. The present invention has found that Aux / IAA This gene can increase the number of plant vascular tissues, construct plant models related to vascular tissue, and cultivate plants with multiple vascular tissues. The first or second SNP site located on this gene is highly significantly correlated with the number of vascular bundles in sisal leaves and can be used to screen for plants with multiple vascular tissues. Therefore, the technical solution provided by the present invention can be applied to the genetic breeding of plant vascular tissue traits, providing a theoretical basis and genetic resources for cultivating new plant varieties or seedlings with increased numbers of vascular columns or vascular bundles. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0018] Figure 1Sisal provided by the present invention Aux / IAA Gene agarose gel electrophoresis verification results; Figure 2 Sisal germplasm resources provided by the present invention Aux / IAA Correlation between the polymorphism of gene 456 and the number of vascular bundles, where ** indicates p <0.01.

[0019] Figure 3 Sisal germplasm resources provided by the present invention Aux / IAA Correlation between the polymorphism of gene locus 602 and the number of vascular bundles, where ** indicates p <0.01.

[0020] Figure 4 Comparison of the wild-type Arabidopsis vascular tissue (A) and the sisal Aux / IAA gene overexpressed in Arabidopsis thaliana (B) provided by the present invention. DETAILED DESCRIPTION

[0021] Unless otherwise specified, the reagents and methods used in the present invention are conventional reagents and methods.

[0022] The present invention provides a sisal Aux / IAA Gene, which was obtained from the sisal transcriptome database, Aux / IAAThe nucleotide sequence of the gene is shown in SEQ ID NO.1 as follows: 5'-ATGGGTGATGCATGTCCAAAGTTGCTTGATTTGATGGTCGCAAAGGAGAGAGATTGGATGGTGAGGGACTCAGGAGGAGGGAGTGGGCTTGGTGTTCAAGAGGAGAAGAAGCTGGAGCTGAAGCTGGGCCCACCAGGAGCTGAGGACTGGGGATCATCAATGGAGAGGAAAGCAGAAGACCCTTCTGCTCTCTCTCTTGGTTACTTCCCTAAAGCCTCTAAAACCACCAAAAGGGGATTTTTAGACACAATTACCTCAAAAAATGAAGGGTTTCAGCAGCAACAGCCTCCTCAGAGCCTTGAGAGGAAGGCATGTTGCCCACCACCATCACATGCTGCTGCTGTCTCTGTTAATGCAGCTAGAATCAATAGCAACAGCTCTCACTCTCAGCCAAGAACTGCTTCTGTTCCAGTAGTTGGGTGGCCTCCCATCCGATCTTTTAGGAAGAATCTCGCGAGCAGCTCCTCCAAGCTGCCACTGGTTGTATCACAGAATGGGGGATCTGAGACTGGAGCAAAGCCTCATATCTGCAAGAAAGGTCTTTTTGTGAAAATTAACATGGATGGGATCCCTATTGGAAGGAAAGTGGACCTCAAGGCCTGTGACAGCTATGAGAAGCTCTCTTCTGCTGTGGGAGAGCTCTTTCAAGGTCTTCTTGCAGCTCAAAAGGATCCAGCTGCTGCTGGAGTTCAGGCCACTGCAGAACAGAAAAAGGCATTTACAGGCTTATTGGATGGGACTGGTGAATACACTTTGGTTTATGACGACAATGAAGGAGACAGGATGCTAGTTGGGGATGTCCCATGGGAAATGTTTGTTTCAACTGCCAAGAGATTGAGGGTGCTGAAGAGCTCTGAACTCTCGACGCTAGCTTTGGGAGCAGTGAGCAGGAAGAGAACAGCAACCGAGTGCCGAGTGGAGTGA-3'.

[0023] The nucleotide sequence of SEQ ID NO. 1 of the present invention contains a SNP site; the SNP site includes: a first SNP site or a second SNP site; the first SNP site is located at the 456th site from the 5' end of the nucleotide sequence of SEQ ID NO. 1, and the site has a G or A mutation; when the first SNP site is A, it is recorded as genotype A, and the nucleotide sequence is as shown in SEQ ID NO.As shown in Figure 7, specifically as follows: 5'-ATGGGTGATGCATGTCCAAAGTTGCTTGATTTGATGGTCGCAAAGGAGAGAGATTGGATGGTGAGGGACTCAGGAGGAGGGAGTGGGCTTGGTGTTCAAGAGGAGAAGAAGCTGGAGCTGAAGCTGGGCCCACCAGGAGCTGAGGACTGGGGATCATCAATGGAGAGGAAAGCAGAAGACCCTTCTGCTCTCTCTCTTGGTTACTTCCCTAAAGCCTCTAAAACCACCAAAAGGGGATTTTTAGACACAATTACCTCAAAAAATGAAGGGTTTCAGCAGCAACAGCCTCCTCAGAGCCTTGAGAGGAAGGCATGTTGCCCACCACCATCACATGCTGCTGCTGTCTCTGTTAATGCAGCTAGAATCAATAGCAACAGCTCTCACTCTCAGCCAAGAACTGCTTCTGTTCCAGTAGTTGGGTGGCCTCCCATCCGATCTTTTAGGAAGAATCTCGCAAGCAGCTCCTCCAAGCTGCCACTGGTTGTATCACAGAATGGGGGATCTGAGACTGGAGCAAAGCCTCATATCTGCAAGAAAGGTCTTTTTGTGAAAATTAACATGGATGGGATCCCTATTGGAAGGAAAGTGGACCTCAAGGCCTGTGACAGCTATGAGAAGCTCTCTTCTGCTGTGGGAGAGCTCTTTCAAGGTCTTCTTGCAGCTCAAAAGGATCCAGCTGCTGCTGGAGTTCAGGCCACTGCAGAACAGAAAAAGGCATTTACAGGCTTATTGGATGGGACTGGTGAATACACTTTGGTTTATGACGACAATGAAGGAGACAGGATGCTAGTTGGGGATGTCCCATGGGAAATGTTTGTTTCAACTGCCAAGAGATTGAGGGTGCTGAAGAGCTCTGAACTCTCGACGCTAGCTTTGGGAGCAGTGAGCAGGAAGAGAACAGCAACCGAGTGCCGAGTGGAGTGA-3'; when the first SNP site is G, it is denoted as the G genotype, and its nucleotide sequence is as shown in SEQ ID NO.1.

[0024] The second SNP site of the present invention is located at the 602nd site from the 5' end of the nucleotide sequence shown in SEQ ID NO.1, and the site contains G or A; when the second SNP site is A, it is recorded as genotype A, and its nucleotide sequence is as shown in SEQ ID NO.As shown in Figure 8, specifically as follows: 5'-ATGGGTGATGCATGTCCAAAGTTGCTTGATTTGATGGTCGCAAAGGAGAGAGATTGGATGGTGAGGGACTCAGGAGGAGGGAGTGGGCTTGGTGTTCAAGAGGAGAAGAAGCTGGAGCTGAAGCTGGGCCCACCAGGAGCTGAGGACTGGGGATCATCAATGGAGAGGAAAGCAGAAGACCCTTCTGCTCTCTCTCTTGGTTACTTCCCTAAAGCCTCTAAAACCACCAAAAGGGGATTTTTAGACACAATTACCTCAAAAAATGAAGGGTTTCAGCAGCAACAGCCTCCTCAGAGCCTTGAGAGGAAGGCATGTTGCCCACCACCATCACATGCTGCTGCTGTCTCTGTTAATGCAGCTAGAATCAATAGCAACAGCTCTCACTCTCAGCCAAGAACTGCTTCTGTTCCAGTAGTTGGGTGGCCTCCCATCCGATCTTTTAGGAAGAATCTCGCGAGCAGCTCCTCCAAGCTGCCACTGGTTGTATCACAGAATGGGGGATCTGAGACTGGAGCAAAGCCTCATATCTGCAAGAAAGGTCTTTTTGTGAAAATTAACATGGATGGGATCCCTATTGGAAGGAAAGTGGACCTCAAGGCCTATGACAGCTATGAGAAGCTCTCTTCTGCTGTGGGAGAGCTCTTTCAAGGTCTTCTTGCAGCTCAAAAGGATCCAGCTGCTGCTGGAGTTCAGGCCACTGCAGAACAGAAAAAGGCATTTACAGGCTTATTGGATGGGACTGGTGAATACACTTTGGTTTATGACGACAATGAAGGAGACAGGATGCTAGTTGGGGATGTCCCATGGGAAATGTTTGTTTCAACTGCCAAGAGATTGAGGGTGCTGAAGAGCTCTGAACTCTCGACGCTAGCTTTGGGAGCAGTGAGCAGGAAGAGAACAGCAACCGAGTGCCGAGTGGAGTGA-3'; when the second SNP site is G, it is denoted as the G genotype, and its nucleotide sequence is as shown in SEQ ID NO.1. The SNP site described in the present invention is extremely significantly correlated with the number of vascular bundles in sisal leaves, that is, the number of vascular bundles in the leaves of sisal seedlings with genotype A is significantly greater than that in the leaves of sisal seedlings with genotype G.

[0025] The present invention provides sisal Aux / IAA The gene encodes the sisal Aux / IAA protein, and the amino acid sequence of the sisal Aux / IAA protein is shown in SEQ ID NO.2, which is specifically as follows: MGDACPKLLDLMVAKERDWMVRDSGGGSGLGVQEEKKLELKLGPPGAEDWGSSMERKAEDPSALSLGYFPKASKTTKRGFLDTITSKNEGFQQQQPPQSLERKACCPPPSHAAAVSVNAARINSNSSHSQPRTASVPVVGWPPIRSFRKNLASSSSKLPLVVSQNGGSETGAKPHICKKGLFVKINMDGIPIGRKVDLKACDSYEKLSSAVGELFQGLLAAQKDPAAAGVQATAEQKKAFTGLLDGTGEYTLVYDDNEGDRMLVGDVPWEMFVSTAKRLRVLKSSELSTLALGAVSRKRTATECRVE*. The SNP site of the present invention is a synonymous mutation, so the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.7 and SEQ ID NO.8 can all encode the sisal Aux / IAA protein with the amino acid sequence shown in SEQ ID NO.2. Aux / IAA The genes and sisal Aux / IAA proteins are related to the vascular tissue traits of plants, and thus the genes or proteins can be used to increase the amount of plant vascular tissue, construct plant models related to vascular tissue, and cultivate and screen plants with multiple vascular tissues.

[0026] Example 1 Sisal Aux / IAA Gene cloning Primers were designed using the nucleotide sequence shown in SEQ ID NO.1 as a template and PCR amplification was performed. The primer sequences are as follows: Forward primer: 5′-ATGGGTGATGCATGTCCAA-3′ (SEQ ID NO. 3); Reverse primer: 5′-TCACTCCACTCGGCACTCGGT-3′ (SEQ ID NO. 4).

[0027] The PCR amplification reaction system (20 μL) is as follows: 1 μL (10 pmol) of forward primer, 1 μL (10 pmol) of reverse primer, 10 μL of EasyTaq® PCR SuperMix, 1 μL of sisal cDNA (template), and 7 μL of ddH2O.

[0028] The PCR amplification reaction program was as follows: denaturation at 95°C for 5 min, 30 cycles of “95°C for 15 s, 60°C for 15 s, and 72°C for 15 s”, and finally 72°C for 2 min.

[0029] The amplified PCR products were subjected to Sanger sequencing, and the electrophoresis results were as follows: Figure 1 As shown (in Figure 1 The left band is the electrophoresis result of the PCR product, and the right band is the marker. The Sanger sequencing results showed that the sequencing results of the PCR product were consistent with the sequence described in SEQ ID NO.1.

[0030] Example 2 Sisal Aux / IAA Verification of the correlation between gene SNP marker sites and the number of vascular bundles in seedlings The primers were designed using the nucleotide sequence shown in SEQ ID NO.1 as a template, and the genome sequences of different sisal germplasm seedlings (all of the above sisal germplasm seedlings were from the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences) were amplified by PCR to analyze the nucleotide sequence shown in SEQ ID NO.1. Aux / IAA Genetic diversity of genes, the primer sequences are as follows: Forward primer: 5′-CCTCCCATCCGATCTTTTAGGAA-3′ (SEQ ID NO. 5); Reverse primer: 5′-GCAAGAAGACCTTGAAAGAGC-3′ (SEQ ID NO. 6).

[0031] The PCR reaction system and reaction procedure were the same as those in Example 1. The template for the PCR reaction was DNA from different sisal germplasm resources.

[0032] Genetic diversity analysis of Aux / IAA genomic sequences amplified from seedlings of different sisal germplasm resources revealed that there were variations at positions 456 or 602 from the 5' end of the nucleotide sequence described in SEQ ID NO.1, with both mutations being G or A. Codon analysis revealed that both positions were synonymous mutations.

[0033] The leaves of different sisal germplasm resources were sliced ​​and the number of vascular bundles was counted under a microscope. The correlation analysis was performed with the two SNP sites. The results are shown in Figure 2 andFigure 3 .

[0034] The results showed that the number of vascular bundles in the leaves of sisal germplasm seedlings of genotype A at position 456 was higher than that of genotype G, and there was a significant difference between the two ( Figure 2 The number of vascular bundles in the leaves of sisal germplasm seedlings of genotype A at position 602 was higher than that of genotype G, and there was a very significant difference between the two ( Figure 3 ).

[0035] The above results show that: Aux / IAA The two SNP sites of the gene are extremely significantly correlated with the number of vascular bundles in sisal seedlings. They can be used to identify the number of vascular bundles in sisal germplasm resources or breeding material seedlings. They have application value in the genetic breeding of sisal vascular bundle traits and provide a theoretical basis for the breeding of new sisal varieties.

[0036] Example 3 Sisal Aux / IAA Application of genes in increasing the number of plant vascular cylinders The sisal hemp obtained by cloning in Example 1 Aux / IAA The gene was introduced into the pEGAD expression vector and introduced into Agrobacterium tumefaciens GV3101 via electroporation. Agrobacterium containing the expression vector was cultured in LB liquid medium with shaking overnight (28°C) until the exponential phase (OD600 value 0.8-1.0). The cells were harvested by centrifugation at 4000 gp / min at room temperature for 5 minutes, transferred to a suspension medium, and suspended to an OD600 value of 0.6. Acetosyringone (AS) (100 μmol / L) was then added for transformation into Arabidopsis thaliana using the floral dip method. Flowering pods of Arabidopsis plants were removed, and the flowers were immersed in the bacterial solution for 60 seconds and left to moisten overnight. Transformation was repeated one week later.

[0037] The seeds of the transformed plants were collected, disinfected with sodium hypochlorite, and inoculated into MS medium containing 50 mg / L kanamycin. After culturing at 22°C ± 2°C for one week, the normally germinated positive plants were selected and planted in soil, cultured and managed, and T1 generation seeds were collected. The T1 generation seeds were planted, and leaves of the T1 generation plants were cut for transgenic plant PCR detection to obtain transgenic positive plants; the transgenic positive plants were cultured and managed, and T2 generation seeds were collected. The transgenic T2 generation seeds and wild-type Arabidopsis seeds were planted in soil respectively and cultivated under the same conditions until the flowering period. The flower axes of the T2 plants and wild-type plants were cut to prepare paraffin sections for histomorphological observation. The results are shown in ( Figure 4 ).

[0038] Combine Figure 4 The results showed that sisal Aux / IAA The number of vascular cylinders in Arabidopsis plants with overexpressed sisal gene increased by 33.33% compared with the wild type, indicating that Aux / IAAGenes can regulate the development of vascular tissue and can be used in genetic breeding of vascular tissue traits to cultivate new plant varieties or seedlings with increased number of vascular columns.

[0039] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Sisal Aux / IAA A gene characterized by The sisal Aux / IAA The gene includes a first SNP site or a second SNP site; The sisal Aux / IAA The nucleotide sequence of the gene includes any one of (a1) to (a3): (a1). As shown in SEQ ID NO.1; (a2). The nucleotide sequence containing the first SNP site is shown in SEQ ID NO. 7; (a3). The nucleotide sequence containing the second SNP site is shown in SEQ ID NO.

8.

2. Sisal according to claim 1 Aux / IAA A gene characterized by In the same plant germplasm, the number of vascular bundles in the leaves of genotype A was significantly greater than that in genotype G. The A genotype is: sisal Aux / IAA The nucleotide sequence of the gene is shown in SEQ ID NO.7 or SEQ ID NO.8; The G genotype is: sisal Aux / IAA The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. Sisal hemp according to claim 1 Aux / IAA The sisal Aux / IAA protein encoded by the gene has an amino acid sequence shown in SEQ ID NO.

2.

4. A biomaterial, characterized in that The biological material includes: containing sisal Aux / IAA Recombinant expression vector or recombinant microorganism of the gene.

5. The sisal hemp according to claim 1 or 2 Aux / IAA Use of the gene, the sisal Aux / IAA protein according to claim 3 or the biomaterial according to claim 4 in plants.

6. The use according to claim 5, characterized in that The plants include one or more of sisal, corn, rice, tobacco and Arabidopsis.

7. The use according to claim 5 or 6, characterized in that The applications include: increasing the amount of plant vascular tissues, constructing plant models related to vascular tissues, and cultivating and screening plants with multiple vascular tissues.

8. The use according to claim 7, characterized in that The plant vascular tissue includes: vascular bundles in leaves and / or vascular columns in flower axes.

9. A method for cultivating multivascular tissue plants, characterized in that: The steps include: Increase the sisal content in plants Aux / IAA The expression level of the gene or the content of the sisal Aux / IAA protein in the plant is increased to obtain a multivascular tissue plant.

10. A method for screening multi-dimensional tubular sisal, characterized in that: The steps include: Using the DNA of the sisal to be tested as a template, PCR amplification and sequencing analysis were performed in sequence; When the nucleotide sequence of the PCR product is as shown in SEQ ID NO.7 or SEQ ID NO.8, the sisal to be tested is multivascular sisal; When the nucleotide sequence of the PCR product is as shown in SEQ ID NO. 1, the plant to be tested is oligovascular sisal.

Images