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Rice Lamina Joint Inclination Assay
水稻叶节夹角测定   

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Abstract

Brassinosteroids (BRs) promote rice lamina inclination. Recently, we showed that OsBUL1 knockout mutant rice (osbul1) is defective in brassinosteroid signaling (Jang et al., 2017). To show that lamina joint inclination of osbul1 is less-sensitive than WT to exogenous brassinolide (BL) treatment in the lamina joint inclination bioassays, we applied the protocol presented below. The protocol focuses on: (1) how to prepare rice samples for the assay, and (2) how to treat BL exogenously. Finally, we have added a result showing lamina inclination between WT and osbul1 in BL solutions of various concentrations.

Keywords: Bioassay(生物测定), Brassinosteroid(油菜素类固醇), Lamina inclination(叶夹角), Lamina joint(叶节), Rice(水稻)

Background

The rice lamina joint connects the leaf blade and sheath, contributing significantly to the leaf angle trait and BR is the main regulator of the trait, while other plant hormones, including ethylene, gibberellin, and auxin, also influence leaf angle (Gan et al., 2015). A more erect leaf facilitates the penetration of sunlight, enhancing photosynthetic efficiency and occupying less space in dense planting (Sakamoto et al., 2006). Thus, rice lamina inclination is one of the major agronomic traits affecting rice plant architecture. Actually, the rice lamina inclination assay developed mainly by Wada and his co-workers is a highly specific and sensitive bioassay for BRs (Wada et al., 1981 and 1984). In this bioassay, treatment with BRs induces greater cell expansion of adaxial cells relative to the abaxial cells in the joint regions, causing laminar inclination in a concentration-dependent manner (Takeno and Pharis, 1982; Cao and Chen, 1995). Changes in cell wall extensibility or loosening are essential for cell expansion (Campbell and Braam, 1999). Although the molecular mechanism for such action remains elusive, cell wall loosening enzymes including xyloglucan endotransglycosylase have been shown to be upregulated by BL and involved in this modification, resulting in laminar inclination in rice (Uozu et al., 2000). Thus, here we describe a procedure through which we could distinguish the BR sensitivity between the wild type and erect leafed osbul1 mutant plants through the rice lamina inclination assay.

Materials and Reagents

  1. 250 µl pipette tips (Mettler-Toledo International, Rainin, catalog number: 17007479 )
  2. 1 ml pipette tips (Mettler-Toledo International, Rainin, catalog number: 17001121 )
  3. 50 ml SuperClear centrifuge tube (Labcon, catalog number: LAB3181 )
  4. Filter paper (Advantec, No.1: 90 mm)
  5. Petri dish, round, 90 x 15 mm (Alpha Plus, catalog number: 16001-1 )
  6. 50 ml syringe (Sigma-Aldrich, catalog number: Z124990 )
  7. Syringe filter (VWR, catalog number: 89041-306 )
  8. Micropore tape (3M, catalog number: 1530-0 )
  9. 1 ml tubes
  10. Rice seeds: Orysa sativa spp. japonica cv. Hwayoung and OsBUL1 knockout mutant rice (osbul1)
  11. Ethanol (Avantor Performance Materials, J.T. Baker®, catalog number: 8006 )
  12. Sodium hypochlorite (NaOCl, Commercial Bleach–CLOROX)
  13. Tween 20 (Alfa Aesar, Affymetrix/USB, catalog number: J20605 )
  14. Potassium hydroxide (KOH) (SHOWA, catalog number: 1637-0150 )
  15. Murashige & Skoog basal medium with Vitamins (MS) (PhytoTechnology Laboratories, catalog number: M519 )
  16. Sucrose (Alfa Aesar, Affymetrix/USB, catalog number: J21938 )
  17. Phytogel (Sigma-Aldrich, catalog number: P8169-500G )
  18. Brassinolide (BL) (Sigma-Aldrich, catalog number: E1641 )
  19. Sodium hypochlorite solution (with final available chlorine of 2%) (see Recipes)
  20. 5 N potassium hydroxide (KOH) solution (see Recipes)
  21. Murashige & Skoog (MS) media (see Recipes)
  22. 1 mM Brassinolide (BL) stock solution (see Recipes)

Equipment

  1. Rice husker (KETT ELECTRIC LABORATORY, model: TR-130 )
  2. Ultrasonic cleaner (Elma, model: E-30H )
  3. Clean bench (Chu-An, model: MBH-420N )
  4. Scissors (Basic Life, catalog number: 76000 )
  5. Forceps (Basic Life, catalog number: BL6502 )
  6. Growth chamber (CHANG KUANG, model: CK-68EX )
  7. Digital camera (Sony, model: NEX-3N )
  8. Protractor (Taiwan united stationery, catalog nunber: HA401 )
  9. 600 ml beaker (DWK Life Sciences, DURAN, catalog number: 21 106 48 )
  10. Glass petri dish (Sun Chion, catalog number: B16A1-0090 )
  11. Autoclave
  12. 10 ml measuring cylinders (DWK Life Sciences, DURAN, catalog number: 21 390 08 04 )
  13. 100 ml measuring cylinders (DWK Life Sciences, DURAN, catalog number: 21 390 24 02 )
  14. 500 ml measuring cylinders (DWK Life Sciences, DURAN, catalog number: 21 390 44 03 )
  15. Vortex mixer (Vortex-Genie2, Scientific Industries, model: Model G560 )
  16. Incubator (YIHDER TECHNOLOGY, model: LM-570RD )
  17. Pipetmans (Gilson, models: P20 , P200 and P1000 )
  18. RiOsTM Essential 16 Water Purification System (EMD Millipore, model: RiOsTM Essential 16 )
  19. Summit Series Analytical Balance (Denver Instrument, model: SI-234 )
  20. pH meter (UltraBasic Benchtop pH Meter, Denver Instrument, model: UB-10 )

Software

  1. ImageJ (https://imagej.nih.gov/ij/) for lamina angle measurement

Procedure

  1. Seedling preparation for lamina inclination
    1. Surface sterilize rice seeds
      1. Remove the lemma and palea of seeds using a rice husker (Figure 1).


        Figure 1. Rice husker used

      2. Put 50-60 naked seeds into a 50 ml SuperClear centrifuge tube and sterilize the surface of the seeds with 30 ml of 70% ethanol for 1 min and vigorously shake by hand.
      3. Rinse the seeds with 30 ml sterile water and pour off the dirty liquid.
      4. Add 30 ml of 2% sodium hypochlorite solution (see Recipes) and place the tube in an ultrasonic cleaner for 20 min.
      5. Discard the sodium hypochlorite solution and wash the seeds with 30 ml sterile water in the clean bench. We usually repeat the washing process 10-15 times.
      6. Place the seeds on autoclaved filter paper to dry and then transfer 20 seeds into a beaker containing MS media (see Recipes) using sterile forceps in the clean bench.
    2. Germinate the sterilized seeds inside a growth chamber under long days (16 h light) at 28 °C for 8 days.
    3. Sample uniform seedlings (based on similar height in each genotype) by excising approximately 2 cm segments (Figure 2) containing the second-leaf lamina joint, leaf blade and leaf sheath and float excised samples on sterile water for 10 min before transferring them to BL solution.


      Figure 2. Eight-day-old rice seedlings for lamina inclination assay. WT (Hwayoung cultivar) and osbul1 mutant rice were grown in beakers containing MS media (left). Leaf segments containing the second-leaf lamina joint of rice seedlings are marked by boxes outlined with dotted lines, which were used for lamina inclination assay.

  2. Exogenous BL treatment
    1. Put 20 ml of each test BL solution at designated concentrations (0 M, 10-6 M, 10-7 M, 10-8 M and 10-9 M in water) in 90 x 15 mm Petri dishes. The solution should be prepared immediately before use.
    2. Float leaf lamina segments on BL solution (see Recipes) (0 M, 10-6 M, 10-7 M, 10-8 M and 10-9 M) in an incubator at 29 °C in the dark for 2 days (Figure 3).


      Figure 3. BR-induced lamina joint inclination in WT (Hwayoung cultivar) and OsBUL1 knock-out mutant (osbul1) rice. The photo was taken after BL (10-6 M) treatment for 2 days.

    3. After 2 days, take sample photos with a digital camera, print them and measure the angle induced between the lamina and the sheath (degree of angle of leaf blade against the axis of leaf sheath) with a protractor (Figure 4).


      Figure 4. Measurement of lamina angles. A. Measuring the lamina angle with a protractor; B. Representative leaf segments containing lamina joint, blade and sheath from WT (Hwayoung cultivar) and OsBUL1 KO (osbul1) plants after incubation for 48 h in the BL solution at different concentrations. C. Reduced leaf angles of OsBUL1 dsRNAi line (#2) compared to WT (TNG67 cultivar) in 0 M, 10-8 M and 10-6 M BL solution (*P < 0.001). 

Data analysis

The same number of rice seedlings was used for each BL concentration set (n = 6-12). When ImageJ software was used for lamina angle measurement, photo files containing rice lamina fragments were opened in the program. By using the angle tool in the software (https://imagej.nih.gov/ij/docs/tools.html), 3 points, one each for the lamina blade, joint and sheath in the lamina fragment were fixed and the angle made by the three points was measured (Figure 5). Measured angle values are presented as means with standard deviations using Microsoft Office 2011 Excel (Figure 4C). The Student’s t-test is used for statistical significance. Data from at least three independent repeats were obtained.


Figure 5. Measurement of lamina angles using the ImageJ program. The three points are marked by green arrows using the angle tool. The measured angle is marked by the box with a dashed red border.

Notes

BL solution should be freshly prepared. Since the sensitivity of this test varied within the rice cultivars employed, crossed data analyses with different rice cultivars should be avoided. For lamina angle measurement, image processing computer programs such as ImageJ can also be used.

Recipes

  1. Sodium hypochlorite solution (with final available chlorine of 2%) for 30 ml
    Add 10 ml of commercial Bleach (CLOROX) into 20 ml of sterile water containing a few drops of Tween 20
  2. 5 N potassium hydroxide (KOH) solution for 30 ml
    Add 8.417g KOH into 30 ml distilled water and filter the solution with a syringe filter
  3. Murashige & Skoog (MS) media for 500 ml
    2.2 g Murashige & Skoog basal medium with Vitamins
    15 g sucrose
    Distilled water up to 500 ml
    Adjust pH to 5.7 with a few drops of 5 N KOH, and distribute 100 ml into a 600 ml beaker containing 0.3 g phytogel each, and then cover the beaker with a glass Petri dish. Seal the beaker with 3M micropore tape and autoclave
  4. 1 mM Brassinolide (BL) stock solution
    1. Dissolve 2 mg of BL (molecular weight 480.68) in 1 ml of ethanol
    2. Add 3.16 ml of distilled water to get 1 mM final concentration
    3. Then split into aliquots in 1 ml tubes and store at -20 °C
    4. Serial dilution: e.g., to make 10-4 M BL solution, 1 ml of BL (10-3 M) mixed with 9 ml of sterile water becomes 10 ml of 10-4 M BL solution

Acknowledgments

This protocol was adapted from the method for rice lamina inclination assay described by Jang et al. (2017). We thank Ms. Miranda Loney for help with English editing. This work was supported in part by a core grant from BCST of ABRC, Academia Sinica.

References

  1. Campbell, P. and Braam, J. (1999). Xyloglucan endotransglycosylases: diversity of genes, enzymes and potential wall-modifying functions. Trends Plant Sci 4(9): 361-366.
  2. Cao, H. and Chen, S. (1995). Brassinosteroid-induced rice lamina joint inclination and its relation to indole-3-acetic acid and ethylene. Plant Growth Regul 16(2): 189-196.
  3. Gan, L., Wu, H., Wu, D., Zhang, Z., Guo, Z., Yang, N., Xia, K., Zhou, X., Oh, K., Matsuoka, M., Ng, D. and Zhu, C. (2015). Methyl jasmonate inhibits lamina joint inclination by repressing brassinosteroid biosynthesis and signaling in rice. Plant Sci 241: 238-245.
  4. Jang, S., An, G. and Li, H. Y. (2017). Rice leaf angle and grain size are affected by the OsBUL1 transcriptional activator complex. Plant Physiol 173(1): 688-702.
  5. Sakamoto, T., Morinaka, Y., Ohnishi, T., Sunohara, H., Fujioka, S., Ueguchi-Tanaka, M., Mizutani, M., Sakata, K., Takatsuto, S., Yoshida, S., Tanaka, H., Kitano, H. and Matsuoka, M. (2006). Erect leaves caused by brassinosteroid deficiency increase biomass production and grain yield in rice. Nat Biotechnol 24(1): 105-109.
  6. Takeno, K. and Pharis, R. P. (1982). Brassinosteroid-induced bending of leaf lamina of dwarf rice seedlings: an auxin-mediated phenomenon. Plant Cell Physiol 23(7): 1275-1281.
  7. Uozu, S., Tanaka-Ueguchi, M., Kitano, H., Hattori, K. and Matsuoka, M. (2000). Characterization of XET-related genes of rice. Plant Physiol 122(3): 853-859.
  8. Wada, K., Marumo, S, Abe, H., Morishita, T., Nakamura, K., Uchiyama, M. and Mori, K. (1984). A rice lamina inclination test–a micro-quantitative bioassay for brassinosteroids. Agric Biol Chem 48(3): 719-726.
  9. Wada, K., Marumo, S., Ikekawa, N., Morisaki, M. and Mori, K. (1981). Brassinolide and homobrassinolide promotion of lamina inclination of rice seedlings. Plant Cell Physiol 22(2): 323-325.

简介

油菜素类固醇(BR)促进水稻倾角。 最近,我们显示,OsBUL1敲除突变体水稻(osbul1 )在油菜素内酯信号传导中有缺陷(Jang等人,2017)。 为了显示在层板关节倾斜生物测定中,osbul1 的椎板关节倾斜对外源油菜素内酯(BL)处理的敏感性低于WT,我们应用了下面提出的方案。 方案重点是:(1)如何准备水稻样品进行测定,以及(2)如何外源性处理BL。 最后,我们在不同浓度的BL溶液中添加了显示WT和osbul1 之间的椎板倾斜度的结果。
【背景】稻叶关节连接叶片和鞘,对叶角特征有显着贡献,BR是性状的主要调节因子,而其他植物激素(包括乙烯,赤霉素和生长素)也影响叶角(Gan et al。,2015)。更直立的叶片有助于阳光的渗透,提高光合作用效率,占据较少的密集种植空间(Sakamoto等人,2006)。因此,稻米倾角是影响水稻植物结构的主要农艺性状之一。实际上,Wada主要开发的稻米倾斜测定法,他的同事们是BR的高度特异性和敏感的生物测定(Wada等人,1981年和1984年)。在这种生物测定中,BR的处理相对于接合区域中的背轴细胞诱导更大的细胞扩增,导致层状倾斜(Takeno和Pharis,1982; Cao和Chen,1995)。细胞壁扩展或松动的变化对于细胞扩增是必需的(Campbell和Braam,1999)。尽管这种作用的分子机制仍然难以捉摸,但已经显示包括木葡聚糖内转葡糖苷酶在内的细胞壁松动酶被BL上调并涉及该修饰,导致水稻层流倾向(Uozu等, 2000)。因此,我们在这里描述了一个程序,通过它们可以通过稻叶倾角测定来区分野生型和直立的叶绿体突变体植物的BR敏感性。

关键字:生物测定, 油菜素类固醇, 叶夹角, 叶节, 水稻

材料和试剂

  1. 250μl移液器吸头(Mettler-Toledo International,Rainin,目录号:17007479)
  2. 1 ml移液器吸头(Mettler-Toledo International,Rainin,目录号:17001121)
  3. 50ml SuperClear离心管(Labcon,目录号:LAB3181)
  4. 滤纸(Advantec,No.1:90 mm)
  5. 培养皿,圆形,90 x 15毫米(Alpha Plus,目录号:16001-1)
  6. 50ml注射器(Sigma-Aldrich,目录号:Z124990)
  7. 注射器过滤器(VWR,目录号:89041-306)
  8. Micropore磁带(3M,目录号:1530-0)
  9. 1毫升管子
  10. 水稻种子:Orysa sativa spp。 cv。 Hwayoung和OsBUL1 敲除突变体水稻( osbul1 )
  11. 乙醇(Avantor Performance Materials,J.T.Baker ®,目录号:8006)
  12. 次氯酸钠(NaOCl,商业漂白剂CLOROX)
  13. 吐温20(Alfa Aesar,Affymetrix / USB,目录号:J20605)
  14. 氢氧化钾(KOH)(SHOWA,目录号:1637-0150)
  15. Murashige&amp;具有维生素(MS)的Skoog基础培养基(PhytoTechnology Laboratories,目录号:M519)
  16. 蔗糖(Alfa Aesar,Affymetrix / USB,目录号:J21938)
  17. 植物凝胶(Sigma-Aldrich,目录号:P8169-500G)
  18. 油菜素内酯(BL)(Sigma-Aldrich,目录号:E1641)
  19. 次氯酸钠溶液(最终有效氯含量为2%)(见配方)
  20. 5 N氢氧化钾(KOH)溶液(见配方)
  21. Murashige&amp; Skoog(MS)媒体(见配方)
  22. 1毫升芥子苷(BL)储备溶液(参见食谱)

设备

  1. 稻壳(KETT ELECTRIC LABORATORY,型号:TR-130)
  2. 超声波清洗机(Elma,型号:E-30H)
  3. 洁净台(Chu-An,型号:MBH-420N)
  4. 剪刀(基本寿命,目录号:76000)
  5. 镊子(基本寿命,目录号:BL6502)
  6. 生长室(长康,型号:CK-68EX)
  7. 数码相机(索尼,型号:NEX-3N)
  8. 量角器(台湾联合文具,产品编号:HA401)
  9. 600ml烧杯(DWK Life Sciences,DURAN,目录号:21 106 48)
  10. 玻璃培养皿(Sun Chion,目录号:B16A1-0090)
  11. 高压灭菌器
  12. 10 ml量筒(DWK Life Sciences,DURAN,目录号:21 390 08 04)
  13. 100ml量筒(DWK Life Sciences,DURAN,目录号:21 390 24 02)
  14. 500 ml量筒(DWK Life Sciences,DURAN,目录号:21 390 44 03)
  15. 涡旋混合器(Vortex-Genie2,Scientific Industries,型号:Model G560)
  16. 孵化器(YIHDER TECHNOLOGY,型号:LM-570RD)
  17. Pipetmans(Gilson,型号:P20,P200和P1000)
  18. 基础16水净化系统(EMD Millipore,型号:RiOs TM Essential 16)
  19. 峰会系列分析天平(丹佛仪器,型号:SI-234)
  20. pH计(UltraBasic台式pH计,丹佛仪器,型号:UB-10)

软件

  1. ImageJ( https://imagej.nih.gov/ij/ )用于层板角度测量

程序

  1. 幼苗倾斜准备
    1. 表面灭菌水稻种子
      1. 使用稻壳去除种子的外a和内ker(图1)

        图1.稻壳使用

      2. 将50-60个裸露的种子放入50 ml SuperClear离心管中,用30 ml的70%乙醇对种子表面灭菌1 min,用手大力摇动。
      3. 用30毫升无菌水冲洗种子,倒掉脏液体。
      4. 加入30 ml的2%次氯酸钠溶液(参见食谱),并将管置于超声波清洗机中20分钟。
      5. 弃去次氯酸钠溶液,并在清洁工作台上用30 ml无菌水冲洗种子。我们通常重复洗涤过程10-15次。
      6. 将种子放在高压灭菌的滤纸上干燥,然后使用无菌镊子将20个种子转移到含有MS培养基的烧杯中(参见食谱)。
    2. 长时间(16小时光照)在28℃下将生长室内的灭菌种子发芽8天。
    3. 通过将含有第二叶片叶片和叶鞘的大约2cm的片段(图2)切除样品均匀的幼苗(基于每个基因型中相似的高度),并将其移至无菌水上10分钟,然后将其转移到BL解决方案

      图2.用于叶片倾斜测定的八日龄水稻幼苗将WT(Hwayoung栽培品种)和osbul1突变体水稻生长在含有MS培养基的烧杯(左)中。含有水稻幼苗第二叶片接头的叶片用虚线标示,用于层板倾斜测定。

  2. 外源性BL治疗
    1. 以指定的浓度(0μM,10μM-6μM,10μM-7μM,10μg-8μM)将20μL各试验BL溶液和10μL-9在水中)在90×15mm培养皿中。解决方案应在使用前立即进行准备。
    2. BL溶液中的浮叶片段(参见食谱)(0M,10 <-6> M,10 <-7> M,10-sup -8和10 -9 M)在29℃的黑暗中孵育2天(图3)。


      图3.在WT(Hwayoung品种)和OsBUL1敲除突变体(osbul1)水稻中BR诱导的椎板关节倾斜。照片是在BL(10 -6 M)治疗2天后服用。

    3. 2天后,用数码相机取样照片,用量角器打印并测量层间和鞘层之间的角度(叶片角度与叶鞘的角度)(图4)。 >

      图4.椎板角度测量。 A.用量角器测量椎板角度; B.在不同的BL溶液中培养48小时后,从WT(Hwayoung品种)和OsBUL1KO(osbul1)植物中分离含有叶片关节,叶片和鞘的代表性叶段浓度。 C.在0μM,10μM-8μM和10μg-6μM中与WT(TNG67栽培品种)相比,降低了OsBUL1 dsRNAi系(#2)的叶角度, / sup> M BL解决方案(* P <0.001)。&nbsp;

数据分析

对于每个BL浓度组(n = 6-12)使用相同数量的水稻秧苗。当ImageJ软件用于层板角度测量时,程序中打开包含米片碎片的照片文件。通过使用软件中的角度工具( https:// imagej .nih.gov / ij / docs / tools.html ),3个点,每个椎板片,椎板片中的关节和鞘均固定,并测量三点制成的角度(图5 )。测量角度值以Microsoft Office 2011 Excel的标准偏差表示(图4C)。 Student's t -test用于统计学意义。获得至少三次独立重复的数据。


图5.使用ImageJ程序测量层板角度使用角度工具用三个点标记绿色箭头。测得的角度由红色边框虚线标记。

笔记

BL解决方案应该做好准备。由于该测试的敏感性在所用水稻品种中不同,因此应避免与不同水稻品种的杂交数据分析。对于层板角度测量,也可以使用诸如ImageJ的图像处理计算机程序。

食谱

  1. 次氯酸钠溶液(最终有效氯为2%)为30 ml
    将10ml商业漂白剂(CLOROX)加入20ml含有几滴吐温20的无菌水中,
  2. 5 N氢氧化钾(KOH)溶液30 ml
    将8.417g KOH加入30ml蒸馏水中,并用注射器过滤器过滤溶液
  3. Murashige&amp; Skoog(MS)介质500 ml
    2.2 g Murashige&amp; Skoog基础培养基维生素
    15克蔗糖
    蒸馏水可达500 ml
    用几滴5N KOH调节pH至5.7,并将100ml分配到含有0.3g植物凝胶的600ml烧杯中,然后用玻璃培养皿覆盖烧杯。用3M微孔带和高压釜密封烧杯
  4. 1mM油菜素甙(BL)储备液
    1. 将2mg BL(分子量480.68)溶于1ml乙醇中
    2. 加入3.16ml蒸馏水,得到1mM终浓度
    3. 然后在1 ml管中分成等分试样并储存于-20°C
    4. 串联稀释:例如,制备10μL/ L M BL溶液,将1ml BL(10-sup-3)与9ml的无菌水变成10毫升10毫升的M BL溶液

致谢

该方案改编自Jang等人描述的水稻层倾角测定方法。 (2017年)。我们感谢米兰达·洛尼女士帮助英语编辑。这项工作部分得到中央研究院ABRC BCST的核心资助。

参考

  1. Campbell,P.和Braam,J。(1999)。葡聚糖内切糖基酶:基因,酶和潜在的壁修复功能的多样性。趋势植物科学 4(9):361-366。
  2. Cao,H。和Chen,S。(1995)。&nbsp; 油菜素类固醇诱导的稻谷关节倾向及其与吲哚-3-乙酸和乙烯的关系。植物生长调节 16(2):189-196。
  3. Gan,L.,Wu,H.,Wu,D.,Zhang,Z.,Guo,Z.,Yang,N.,Xia,K.,Zhou,X.,Oh,K.,Matsuoka, Ng,D. and Zhu,C.(2015)。&nbsp; 茉莉酸甲酯通过抑制水稻中的油菜素类固醇生物合成和信号传导来抑制层状关节倾向。植物科学241:238-245。
  4. Jang,S.,An,G.and Li,HY(2017)。&nbsp; 水稻叶角度和粒度受OsBUL1转录激活子复合物的影响。植物生理学173(1):688-702。
  5. Sakamoto,T.,Morinaka,Y.,Ohnishi,T.,Sunohara,H.,Fujioka,S.,Ueguchi-Tanaka,M.,Mizutani,M.,Sakata,K.,Takatsuto,S.,Yoshida,S 。,Tanaka,H.,Kitano,H.and Matsuoka,M。(2006)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/ 16369540“target =”_ blank“>由油菜素类固醇缺乏导致的直立叶增加了水稻的生物量产量和籽粒产量。生物技术 24(1):105-109。
  6. Takeno,K.和Pharis,RP(1982)。芸苔素类固醇诱导的矮秆幼苗叶片弯曲:生长素介导的现象。植物细胞生理学23(7):1275-1281。
  7. Uozu,S.,Tanaka-Ueguchi,M.,Kitano,H.,Hattori,K.and Matsuoka,M。(2000)。&lt; a class =“ke-insertfile”href =“http: ncbi.nlm.nih.gov/pubmed/10712549“target =”_ blank“>水稻XET相关基因的表征。植物生理学122(3):853-859。 br />
  8. Wada,K.,Marumo,S,Abe,H.,Morishita,T.,Nakamura,K.,Uchiyama,M.and Mori,K。(1984)。&lt; a class =“ke-insertfile”href = “https://www.jstage.jst.go.jp/article/bbb1961/48/3/48_3_719/_article”target =“_ blank”>水稻倾角测试 - 油菜素类固醇的微量子生物测定。农业生物化学 48(3):719-726。
  9. Wada,K.,Marumo,S.,Ikekawa,N.,Morisaki,M.and Mori,K。(1981)。&lt; a class =“ke-insertfile”href =“http://agris.fao。组织/ agris-search / search.do?recordID = JP8204687“target =”_ blank“>油菜素内酯和高芥子油素促进水稻幼苗的叶片倾向。植物细胞生理学22(2): 323-325。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Li, H., Wang, H. and Jang, S. (2017). Rice Lamina Joint Inclination Assay. Bio-protocol 7(14): e2409. DOI: 10.21769/BioProtoc.2409.
  2. Jang, S., An, G. and Li, H. Y. (2017). Rice Leaf Angle and Grain Size Are Affected by the OsBUL1 Transcriptional Activator Complex. Plant Physiol 173(1): 688-702.
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