Construction method and application of cyclocarya paliurus leaf VIGS instantaneous silencing system

Abstract

The invention provides a construction method of a cyclocarya paliurus leaf VIGS instantaneous silencing system. The construction method comprises the following steps: connecting a CpPDS gene with pTRV2 to obtain a vector pTRV2-CpPDS; the method comprises the following steps: respectively transforming carriers pTRV2-CpPDS and pTRV1 into agrobacterium tumefaciens, and then coating an LB solid culture medium; the method comprises the following steps: respectively inoculating agrobacterium containing a carrier pTRV2-CpPDS and agrobacterium containing a carrier pTRV1 into an LB liquid culture medium; collecting thalli, and resuspending the precipitate by using an infection solution; mixing the two resuspension solutions to obtain a dip dyeing bacterial solution; the leaf backs and leaf stems of the cyclocarya paliurus are scratched, and the cyclocarya paliurus are put into the dip-dyeing bacterial liquid to be soaked, or the scratched parts are wrapped with cotton absorbing the dip-dyeing bacterial liquid. The invention also provides application of the method in verification of cyclocarya paliurus gene functions. Cyclocarya paliurus leaves are infected through the method, a silence system of a target gene is constructed, the expression level of the target gene can be reduced, and a foundation is laid for cyclocarya paliurus gene function research.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a method for constructing and applying a transient VIGS silencing system for Cyclocarya paliurus leaves. Background Technology

[0002] Qingqianliu [ Cyclocarya paliurus (Batal) Iljinsk [Juglandaceae family ( Jμglandaceae Cyclocarya genus ( Cyclocarya *Cyclocarya paliurus* is a monotypic genus endemic to my country. The tree is tall, and its fruit, resembling a coin, hangs on the tree for a long period, making it highly ornamental. Since the 1980s, research on the chemical composition and pharmacological activities of *Cyclocarya paliurus* has become a hot topic. Modern pharmacological studies have also shown that *Cyclocarya paliurus* secondary metabolites possess pharmacological activities such as lowering blood sugar, lowering blood lipids, lowering blood pressure, antioxidation, and antitumor effects. With a deeper understanding of the medicinal and health-promoting value of *Cyclocarya paliurus*, market demand is constantly increasing. However, its quality formation mechanism remains unclear, and gene function analysis has not been conducted in many studies. Therefore, establishing a rapid and efficient gene function verification technology for *Cyclocarya paliurus* is currently a key issue in *Cyclocarya paliurus* gene function research.

[0003] Virus-induced gene silencing (VIGS) is an important genomics tool that utilizes the plant's homology-dependent defense mechanism to specifically reduce the expression of endogenous genes in the host. It can rapidly induce the generation of gene-silencing phenotypes in plants, characterizing gene function and providing an efficient and feasible alternative for gene function identification in plants lacking genetic transformation systems. VIGS primarily utilizes viral vectors to carry the target gene and infect plants. During replication and expression, double-stranded RNA (dsRNA) is formed. The dsRNA is cleaved in the cell by the specific endonuclease Dicer into small interfering RNA (siRNA) fragments of 21-24 nt. The siRNA is amplified by RNA polymerase and, in single-stranded form, binds to certain proteins to form RNA-induced silencisCng complexes (RISC). These complexes specifically interact with the target gene mRNA, leading to its degradation and thus post-transcriptional gene silencing. Currently, although VIGS has been applied in many plants, its application and research in *Cyclocarya paliurus* leaves are relatively limited. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a method for constructing and applying a transient VIGS silencing system for Cyclocarya paliurus leaves, in order to lay the foundation for further verification of Cyclocarya paliurus gene function.

[0005] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A method for constructing a transient VIGS silencing system from Cyclocarya paliurus leaves includes the following steps: (1) Qingqianliu CpPDS The gene fragment was ligated into the tobacco brittle virus vector pTRV2 to obtain the recombinant vector pTRV2- CpPDS ; (2) The recombinant vector pTRV2- CpPDS The tobacco brittle virus vector pTRV1 was transformed into Agrobacterium GV3101 to obtain the recombinant vector pTRV2-. CpPDS Agrobacterium and Agrobacterium containing the helper vector pTRV1 were used. The transformed Agrobacterium was spread on LB solid medium. After the colonies grew, single colonies were picked for PCR verification. (3) The pTRV2-- CpPDS Single clones of Agrobacterium and Agrobacterium strain containing the helper vector pTRV1 were inoculated into LB liquid medium for propagation; the bacterial cells were collected by centrifugation, and the corresponding precipitates were resuspended in the infection solution and the OD was adjusted. 600 To 0.6~1.0; (4) Mix the two resuspensions obtained in step (3) in a 1:1 ratio, let stand for 3 hours or shake for 1 hour to obtain the infiltrated bacterial solution; (5) Scratch the back of the leaves of Cyclocarya paliurus and soak the scratched leaves in the inoculum solution obtained in step (4) for 30-60 seconds, or wrap the scratched area with cotton soaked in the inoculum solution to infect the leaves and achieve the silencing of the target gene in Cyclocarya paliurus leaves.

[0006] As one of the preferred embodiments of the present invention, in step (1), *Cyclocarya paliurus* is used. CpPDS The nucleotide sequence of the gene fragment is shown in SEQ ID NO.1.

[0007] As one of the preferred embodiments of the present invention, in step (1), Qian Liu CpPDS The gene fragment was obtained by reverse transcription of RNA extracted from Cyclocarya paliurus leaves as a cDNA template, followed by amplification using KOD high-fidelity enzyme; the amplification primers were: CpPDS -F、 CpPDS -R, the sequences are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively; the amplification program is: 95℃, 3min; 95℃ 15sec, 60℃ 15sec, 72℃ 30sec, 40 cycles; 72℃ 5min for complete extension.

[0008] As one of the preferred embodiments of the present invention, in step (2), the solid culture medium is LB solid culture medium containing 50 μg / mL Kan and 25 μg / mL Rif.

[0009] As one of the preferred embodiments of the present invention, in step (3), the liquid culture medium is LB liquid culture medium containing 50 μg / mL Kan and 25 μg / mL Rif.

[0010] As one of the preferred embodiments of the present invention, in step (3), the formulation of the infiltration solution is: 10mM MgCl2, 10mM MMES, 200μM acetylsuccinone.

[0011] As one of the preferred embodiments of the present invention, in step (5), the infected parts of the Cyclocarya paliurus leaves are the back of the leaves and the leaf stems; and when the infected part is specifically the back of the leaves, the infected part is soaked in the inoculum solution; when the infected part is specifically the leaf stems, the infected part is wrapped with cotton soaked in the inoculum solution.

[0012] As one of the preferred methods of the present invention, in step (5), after the infection is completed, the Cyclocarya paliurus plants are cultured in the dark for 2 days, and then cultured under the cycle of "24℃, 16h light, 20℃, 8h darkness", and watered regularly during the period.

[0013] As one of the preferred embodiments of the present invention, steps (6) and (7) are also included. Step (6): Observe the leaves of the cultured and transformed plants using the photobleaching method. The appearance of white leaves indicates a silent system. Step (7): Collect leaves from *Cyclocarya paliurus* plants, extract RNA, and reverse transcribe it into cDNA as a template for qPCR analysis; and use *Cyclocarya paliurus* as a template for analysis. Cp18S Primers were designed using genes as internal reference genes for [the purpose of] [the study ... CpPDS Genes were analyzed by relative quantitative qRT-PCR.

[0014] As one of the preferred embodiments of the present invention, the Cp18S Primers designed using genes as internal reference genes are Cp18S -F、 Cp18S -R, the sequences are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively.

[0015] As one of the preferred methods of the present invention, the reaction program for qRT-PCR is as follows: 95°C, 2 min; 95°C, 10 sec, 40 cycles; 60°C, 30 s.

[0016] An application of the above-mentioned construction method in verifying the function of the Cyclocarya paliurus gene.

[0017] Design concept and principle of this invention: Tobacco brittle virus-induced gene silencing is currently the most widely used and effective VIGS virus vector in plants, with good infection effects on plants including tomatoes, tobacco, and peppers. However, its infection efficiency on Eucommia ulmoides is still unknown. This invention attempts to conduct VIGS experiments on Eucommia ulmoides based on this vector.

[0018] At the same time, the phytopene dehydrogenase gene ( PDS ( ), is an enzyme required in the carotenoid synthesis pathway; experiments have shown that when PDS When a gene is successfully silenced in tobacco, the plant will exhibit obvious albinism; because the phenotypic changes after gene silencing are significant and easily observed, it can be used to... PDS The gene is a reporter gene used to evaluate whether the VIGS system induces effective silencing in different crops.

[0019] In summary, this invention utilizes the *Cyclocarya paliurus* phytorepinephrine dehydrogenase gene. CpPDS As a positive reporter gene, a VIGS system based on *Cyclocarya paliurus* leaves induced by tobacco brittleness virus was constructed.

[0020] The advantages of this invention compared to the prior art are: (1) This invention utilizes Tobacco Crack Virus (TRV)-mediated verification of the gene function of Eucommia ulmoides, involving the use of TRV viral vectors to verify the gene function of Eucommia ulmoides. PDS The gene marker is a marker gene, which is used in Cyclocarya paliurus leaves through the TRV virus-mediated VIGS system to facilitate subsequent gene function verification and analysis. (2) This invention has the advantages of high efficiency, short cycle, simple operation and economic benefits. The method used does not require the establishment of a genetic transformation system, in order to construct the Cyclocarya paliurus VIGS system and lay the foundation for the study of the gene function of Cyclocarya paliurus. Attached Figure Description

[0021] Figure 1 These are phenotypic images of plants under photobleaching in Example 1 (in the figures, A shows the phenotypic of the silent system with white leaves, and B shows the phenotypic of the blank control plants). Figure 2 These are the qRT-PCR detection results from Example 1 (with wild-type WT and empty vector as controls); Figure 3 Different OD values ​​in Experiment Example 1 600 The effect of the value of the infecting bacterial solution on the silencing efficiency of different parts of Eucommia ulmoides infection (Figure A shows the OD value). 600 0.6 Results of bacterial inoculation; Figure B shows OD. 600 0.8 Results of bacterial inoculation; Figure C shows OD. 600 1.0 Results of bacterial infection ( ). Detailed Implementation

[0022] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents and experimental methods used in the present invention are all conventional reagents or methods in the art and will not be described further.

[0023] Example 1 The method for constructing a transient VIGS silencing system for Cyclocarya paliurus leaves according to this embodiment includes the following steps: (1) Construction of recombinant carrier For the target plant, Cyclocarya paliurus CpPDS The gene-specific fragment (SEQ ID NO.1) was cloned by reverse transcription of RNA extracted from Cyclocarya paliurus leaves as a cDNA template, followed by amplification using KOD high-fidelity enzyme; the amplification primers were as follows. CpPDS -F (SEQ ID NO.2) CpPDS -R (SEQ ID NO.3); the amplification program was: 95℃, 3 min; 95℃ 15 sec, 60℃ 15 sec, 72℃ 30 sec, 40 cycles; 72℃ 5 min for complete extension.

[0024] target gene CpPDS The tobacco brittle virus vector pTRV2 was digested with restriction endonucleases BamHI and XhoI. The double digestion system is shown in Table 1.

[0025] Table 1 Double enzyme digestion system

[0026] After double enzyme digestion, the DNA fragments were detected by agarose gel electrophoresis, and the corresponding fragments were recovered from the gel for DNA purification. Then, they were ligated using ligase to obtain the recombinant vector pTRV2- CpPDS After sequencing verification confirms correctness, it is ready for use.

[0027] (2) Agrobacterium transformation The recombinant vector pTRV2- CpPDS The tobacco brittle virus vector pTRV1 was transformed into Agrobacterium GV3101 to obtain the recombinant vector pTRV2-. CpPDS Agrobacterium and Agrobacterium containing the helper vector pTRV1 were used. The transformed Agrobacterium was plated on LB solid medium containing 50 μg / mL Kan and 25 μg / mL Rif, respectively. After the colonies grew, single colonies were picked for PCR verification.

[0028] (3) Obtaining the resuspension The sequenced correctly contains the recombinant vector pTRV2- CpPDS Single clones of *Agrobacterium* and *Agrobacterium* strains containing the helper vector pTRV1 were inoculated into LB liquid medium containing 50 μg / mL Kan and 25 μg / mL Rif, respectively, and cultured at 28 °C. The cells were collected by centrifugation at 5000 rpm for 10 min, and the precipitates were resuspended in infection buffer (10 mM MgCl2, 10 mM MES, 200 μM acetylsylcholine) and the OD was adjusted. 600 Up to 0.6.

[0029] (4) Preparation of inoculated bacterial solution The two resuspensions obtained in step (3) were mixed in a 1:1 ratio and allowed to stand for 3 hours to obtain "pTRV1+pTRV2- CpPDS "Immersion solution."

[0030] (5) Agrobacterium infection of Cyclocarya paliurus leaves Scratch the underside of the leaves of *Cyclocarya paliurus* and place the scratched leaves into "pTRV1+pTRV2- CpPDS "The plants were immersed in the bacterial solution for 60 seconds for infection (with the "pTRV1+pTRV2" infection solution as a blank control); after infection, the Cyclocarya paliurus plants were cultured in the dark for 2 days, and then cultured under the cycle of "24℃, 16h light, 20℃, 8h darkness", with regular watering during the period.

[0031] (6) Observing plant morphological changes using photobleaching method The leaves of transformed plants cultured for 1-6 weeks were observed using the photobleaching method. The presence of albino leaves indicated a silent system. Figure 1 As shown.

[0032] (7) qRT-PCR detection of whether the target gene is silenced Leaves of *Cyclocarya paliurus* plants were collected on days 2, 4, 8, 16, 24, and 32 after infection completion (as test samples). RNA was extracted, reverse transcribed into cDNA, and used as a template for qPCR analysis. Cp18s Genes as internal reference genes for primer design Cp18S -F (SEQ ID NO.4) Cp18S -R (SEQ ID NO.5), for samples treated with different methods CpPDS Genes were analyzed by relative quantitative qRT-PCR. The qRT-PCR reaction program was: 95℃, 2 min; 95℃, 10 sec, 40 cycles; 60℃, 30 s. The results are as follows: Figure 2 As shown.

[0033] Depend on Figure 1 , Figure 2The results show that the silencing system constructed in this invention can effectively suppress... CpPDS Gene expression.

[0034] Example 2 The method for constructing a transient VIGS silencing system for Cyclocarya paliurus leaves according to this embodiment includes the following steps: (1) Construction of recombinant carrier Same as Example 1.

[0035] (2) Agrobacterium transformation Same as Example 1.

[0036] (3) Obtaining the resuspension The sequenced correctly contains the recombinant vector pTRV2- CpPDS Single clones of *Agrobacterium* and *Agrobacterium* strains containing the helper vector pTRV1 were inoculated into LB liquid medium containing 50 μg / mL Kan and 25 μg / mL Rif, respectively, and cultured at 28 °C. The cells were collected by centrifugation at 5000 rpm for 10 min, and the precipitates were resuspended in infection buffer (10 mM MgCl2, 10 mM MES, 200 μM acetylsylcholine) and the OD was adjusted. 600 Up to 0.8.

[0037] (4) Preparation of inoculated bacterial solution The two resuspended solutions obtained in step (3) were mixed in a 1:1 ratio and shaken for 1 hour to obtain "pTRV1+pTRV2- CpPDS "Immersion solution."

[0038] (5) Agrobacterium infection of Cyclocarya paliurus leaves Scratching the stems and leaves of *Cephalotaxus fortunei* and applying a solution containing "pTRV1+pTRV2-" CpPDS "Cotton soaked in bacterial solution was used to cover the scratched area for infection (with "pTRV1+pTRV2" infection solution as a blank control); after infection, the Cyclocarya paliurus plants were cultured in the dark for 2 days, and then cultured under the cycle of "24℃, 16h light, 20℃, 8h darkness", with regular watering during the period.

[0039] (6) Observing plant morphological changes using photobleaching method Same as Example 1.

[0040] (7) qRT-PCR detection of whether the target gene is silenced Same as Example 1.

[0041] Example 3 The method for constructing a transient VIGS silencing system for Cyclocarya paliurus leaves according to this embodiment includes the following steps: (1) Construction of recombinant carrier Same as Example 1.

[0042] (2) Agrobacterium transformation Same as Example 1.

[0043] (3) Obtaining the resuspension The sequenced correctly contains the recombinant vector pTRV2- CpPDS Single clones of *Agrobacterium* and *Agrobacterium* strains containing the helper vector pTRV1 were inoculated into LB liquid medium containing 50 μg / mL Kan and 25 μg / mL Rif, respectively, and cultured at 28 °C. The cells were collected by centrifugation at 5000 rpm for 10 min, and the precipitates were resuspended in infection buffer (10 mM MgCl2, 10 mM MES, 200 μM acetylsylcholine) and the OD was adjusted. 600 Up to version 1.0.

[0044] (4) Preparation of inoculated bacterial solution The two resuspensions obtained in step (3) were mixed in a 1:1 ratio and allowed to stand for 3 hours to obtain "pTRV1+pTRV2- CpPDS "Immersion solution."

[0045] (5) Agrobacterium infection of Cyclocarya paliurus leaves Scratching the leaf buds of *Cephalotaxus fortunei* and applying a solution containing "pTRV1+pTRV2-" CpPDS "Cotton soaked in bacterial solution was used to cover the scratched area for infection (with "pTRV1+pTRV2" infection solution as a blank control); after infection, the Cyclocarya paliurus plants were cultured in the dark for 2 days, and then cultured under the cycle of "24℃, 16h light, 20℃, 8h darkness", with regular watering during the period.

[0046] (6) Observing plant morphological changes using photobleaching method Same as Example 1.

[0047] (7) qRT-PCR detection of whether the target gene is silenced Same as Example 1.

[0048] Experimental Example 1 This experimental example is used to verify different ODs 600 The effect of the value of the infecting bacterial solution on the silencing efficiency of different parts of Cyclocarya paliurus infection.

[0049] I. Experimental Methods The method is the same as in Example 1, but the OD of the infection solution is adjusted during the preparation of the bacterial inoculum. 600The concentrations of the inoculum were adjusted to 0.6, 0.8, and 1.0, respectively, and the inoculum was applied to the underside of the leaf and the leaf stem. (When the inoculum was applied to the underside of the leaf, it was applied by soaking the inoculum in the inoculum solution; when the inoculum was applied to the leaf stem, it was applied by wrapping the inoculum in cotton soaked in the inoculum solution.) The effects of different concentrations of inoculum on the silencing efficiency of each inoculum were then studied.

[0050] Silencing efficiency = Empty vector injected gene expression level / Contains CpPDS Gene expression level *100% when injected with gene-infecting solution.

[0051] II. Experimental Results The results are shown in Tables 2, 3, and 4. Figure 3 As shown.

[0052] Table 2 OD 600 0.6 Effect of bacterial inoculation on the underside of leaves and leaf stems on the silencing efficiency of Cyclocarya paliurus plants

[0053] Table 3 OD 600 0.8 Effect of bacterial inoculation on the underside of leaves and leaf stalks on the silencing efficiency of Cyclocarya paliurus plants

[0054] Table 4 OD 600 1.0 Effect of bacterial inoculation on the underside of leaves and leaf stalks on the silencing efficiency of Cyclocarya paliurus plants

[0055] The results above show that the OD of the inoculum is... 600 0.6 achieved maximum silencing efficiency of 82.2% at 4 days; the OD of the inoculum... 600 0.8 achieved maximum silencing efficiency of 75.1% at 4 days; OD of the inoculum 600 1.0 achieved its maximum silencing efficiency of 78.8% at 16 days. However, the OD of the inoculum... 600 The silencing efficiency of version 1.0 lasts the longest.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a Cyclobalanopsis glauca leaf VIGS transient silencing system, characterized in that, Comprising the following steps: (1) The C. sinensis CpPDS The C. sinensis CpPDS ; (2) Transform the recombinant vector pTRV2- CpPDS and the tobacco rattle virus vector pTRV1 into Agrobacterium GV3101 respectively to obtain Agrobacterium containing the recombinant vector pTRV2- CpPDS and Agrobacterium containing the auxiliary vector pTRV1; after the transformed Agrobacterium is plated on solid medium and colonies grow, single colonies are picked for PCR verification; (3) the Agrobacterium containing the recombinant vector pTRV2- CpPDS 600 was inoculated into liquid medium for expansion culture; the bacterial cells were collected by centrifugation, and the corresponding precipitate was resuspended with the infection liquid, and the OD​​ (4) Mixing the two resuspensions obtained in step (3) in a ratio of 1:1 to obtain a bacterial liquid for immersion; (5) Scratching the back of the leaf of Cyclobalanopsis glauca, and placing the scratched leaf in the bacterial liquid for immersion obtained in step (4) for immersion for 30-60s, or wrapping the scratched part with cotton absorbing the bacterial liquid for immersion to perform infection, so as to realize silencing of the target gene in the leaf of Cyclobalanopsis glauca.

2. The method according to claim 1, wherein the VIGS transient silencing system of Adenanthera pavonina leaf is constructed by, In step (1), the Cyclocarya CpPDS The nucleotide sequence of the gene fragment is shown as SEQ ID NO.

1.

3. The method according to claim 1, wherein the VIGS transient silencing system of Adenanthera pavonina leaf is constructed by, In the step (1), the adhesion of the adhesion layer to the seed layer is performed by using a solution of the adhesion agent in a solvent, and the adhesion agent is a solution of the adhesion agent in a solvent CpPDS The gene fragment was extracted from the cDNA template of the adhesion agent in a solvent, and the adhesion agent was a solution of the adhesion agent in a solvent CpPDS -F, CpPDS -R, and the sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively; the amplification program is: 95℃, 3min; 95℃ 15sec, 60℃ 15sec, 72℃ 30sec, 40 cycles; 72℃ 5min thorough extension.

4. The method according to claim 1, wherein the VIGS transient silencing system of Adenanthera pavonina leaf is constructed by, In the step (2), the solid culture medium is LB solid culture medium containing 50 μg / mL Kan and 25 μg / mL Rif.

5. The method according to claim 1, wherein the VIGS transient silencing system of Adenanthera pavonina leaf is constructed by, In the step (3), the liquid culture medium is LB liquid culture medium containing 50 μg / mL Kan and 25 μg / mL Rif.

6. The method according to claim 1, wherein the VIGS transient silencing system of Adenanthera pavonina leaf is constructed by, In the step (3), the formula of the infection liquid is: 10 mM MgCl2, 10 mM MES, 200 μM acetyl-syringone.

7. The method according to claim 1, wherein the VIGS transient silencing system is constructed by using Adenocaulon minus VIGS vector pCVD 1280. In the step (5), the infection part of the leaf of Cyclobalanopsis glauca is the back of the leaf and the petiole; and when the infection part is specifically the back of the leaf, the immersion is performed by the method of immersion in the bacterial liquid for immersion; and when the infection part is specifically the petiole, the immersion is performed by the method of wrapping with cotton absorbing the bacterial liquid for immersion.

8. The method according to claim 1, wherein the VIGS transient silencing system of Adenanthera pavonina leaf is constructed by, In the step (5), after the infection is completed, the Cyclobalanopsis glauca plant is cultured in darkness for 2d, and then is cultured under the cycle of "24℃, 16h light, 20℃, 8h darkness", and water is poured regularly during the period.

9. The method according to claim 1, wherein the VIGS transient silencing system of Adenanthera pavonina leaf is constructed by, Further comprising steps (6) and (7); Step (6): observing the leaf of the cultured transformed plant by using photobleaching method, and the appearance of white leaf is the silencing system; Step (7): Collecting the leaves of Cyclocarya paliurus plant, extracting RNA, and reverse transcribing into cDNA as the template for qPCR analysis; and taking the Cyclocarya paliurus Cp18S gene as the internal reference gene to design primers, and performing relative quantitative qRT-PCR analysis on the CpPDS gene.

10. Use of the construction method according to any one of claims 1-9 in verifying the gene function of Cyclobalanopsis glauca.

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