1. The document discusses a study on microRNAs (miRNAs) in Oryza rufipogon, a wild rice species, under drought stress conditions. 2. Small RNA sequencing was performed on shoot and root tissues with or without drought treatment. Differentially expressed miRNAs were identified, including 31 miRNAs regulated in both tissues. 3. The study identified 200 drought-responsive miRNAs and 97 novel miRNAs. Hierarchical clustering analysis showed different expression patterns of miRNAs between shoot and root tissues under drought.

2. “mi RNAs: Small RNAs with an important
role in gene regulation in crop plants.”
SPOORTHI V.
PALB 7077
II Ph.D. (GPB)
GKVK, UAS,
BENGALURU
Seminar-1
on
2

3. RNA AND ITS TYPES
GENE REGULATION AND METHODS
Contents
CASE STUDIES
RNAi AND ITS APPROCHES
mi RNA-BIOGENESIS,TARGET,EXTRACTION, EDITING
DIFFERENCE BETWEEN mi AND siRNA?
CONCLUSION
mi RNA DATA BASES
3

4. Gene regulation
• Gene regulation refers to the mechanisms
that act to induce or repress
the expression of a gene.
4

5. #
Prokaryotes Eukaryotes
Structure of
genome
Single, generally circular,
sometimes accompanied by
smaller pieces of DNA,
plasmid
Genome found in
chromosome,
nucleosome structure
limits DNA
accessibility
Size of genome Relatively small Relatively large
DNA structure Highly super coiled with
some associated protein
Highly super coiled
chromatin, histones in
nucleosomes
Difference Between Prokaryote And Eukaryote Gene
Expression And Regulation

6. Levels of gene regulation
6

7. Different mechanisms of gene regulation
 DNA methylation
 Hormonal control
 RNA interference (siRNA, miRNA)
(Sanghera et al., 2010)
7

8. RNA(Ribonucleic acid)
• Ribonucleic acid
– Ribonucleotides (Ribose, base, & phosphate)
• Types
Coding: messenger RNA (mRNA)
Non-coding: (ncRNA) is a functional RNA molecule that
is not translated into a protein
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
Small nuclear RNA (snRNA)
Small nucleolar RNA (snoRNA)
Interference RNA (RNAi)
(Sanghera et al., 2010)
8

9. mRNA Structure
• Coding region
• Untranslated regions
– 5’ UTR
• 7-methyl-G cap
–Bound by cap binding proteins
• Translation regulation
– 3’ UTR
• Stability elements
• Sub-cellular localization (zip codes)
• poly(A) tail
(Sanghera et al., 2010) 9

10. mRNA
(Sanghera et al., 2010)
10

11. #
RNA interference
(= co-suppression, quelling)
• Diverse RNA-based processes that all result in
sequence-specific inhibition of gene expression
either at the transcription mRNA stability or
translational levels.
• It has most likely evolved as a mechanism for cells to
eliminate foreign genes.
(Sanghera et al., 2010)

12. Discovery
 The silencing effect was first observed in plants in 1990,
when the Jorgensen laboratory introduced exogenous
transgenes into petunias in an attempt to up-regulate the
activity of a gene for chalcone synthase, an enzyme involved
in the production of specific pigments.
 Unexpectedly, flower pigmentation did not deepen, but
rather showed variegation with complete loss of color in
some cases.
 This indicated that not only were the introduced trangenes
themselves inactive, but that the added DNA sequences also
affected expression of the endogenous loci.
– This phenomenon was referred to as “co-suppression”. 12

13. Approaches to induce RNAi
transcriptional levels
Genes
post-transcriptional levels.
• Transcriptional gene silencing:
13

14. • Post-transcriptional gene silencing is the result of
mRNA of a particular gene being destroyed or blocked.
• The destruction of the mRNA prevents translation to
form an active gene product (in most cases, a protein).
• A common mechanism of post-transcriptional gene
silencing is RNAi.
mRNA
transcript/
ribosome
14

15. Block of primary transcription
X
M M M
M M
Promoter methylation
MMM
M M
Post-transcriptional
gene silencing (PTGS)
BL BR
Promoter Coding Region pA
Transcriptional
gene silencing (TGS)
BL BR
Promoter Coding Region pA
mRNA degradation
BL BR
+
M M M
M M
Methylation of the transcribed region
15

16. • Transcriptional gene silencing (TGS)
– Inactivation of (trans)gene-specific nuclear RNA
synthesis.
– Induced by a DNA-mediated or RNA-mediated DNA
methylation.
 methylation of promoter => reduced transcription.
– Post-transcriptional gene silencing (PTGS)
– Reduction in steady-state mRNA levels without
affecting the nuclear transcription
 Normal transcription followed by RNA degradation
As a result of the two mechanisms of gene-silencing,
methylation and / or RNA degradation of specific DNA
/ RNA sequences occurs.
16

17. #

18. #
• Two types of small RNA molecules –
microRNA (miRNA) and
small interfering RNA(siRNA)
are central to RNA
interference

19. miRNA (Micro RNA)
 Single-stranded RNA (20-25 nt long).
 Comparable -transcription factors or RNA-binding proteins
(RBPs).
 Expressed in a tissue-specific or developmental stage-
specific manner.
 It is thought to regulate the expression of other genes.

20. History
20
• lin-4, first miRNA to be described in C. elegans; important in
development of the worm from larva to adult.
• let-7, was also described in C. elegans (Reinhard et al, 2000)
• 1998-Fire and Mello, experiments in C. elegans, first to show
that dsRNA is much more potent at inhibiting gene expression
than antisense RNA. Set the stage for understanding the role of
miRNAs in development and gene regulation. (Nobel Prize in
Physiology and Medicine, 2007).

21. Biogenesis
21

22. Summary of proteins Involved
Drosha and Pasha are part of the “Microprocessor” protein
complex (~600-650kDa).
Drosha and Dicer are RNase III enzymes.
Pasha is a dsRNA binding protein.
Exportin 5 is a member of the karyopherin nucleocytoplasmic
transport factors that requires Ran and GTP.
Argonautes are Rnase H enzymes- needed for miRNA induced
silencing, contain two domains PAZ and PIWI –cleave target
mRNAs in the middle of the site complementary to miRNA.
22

23. • In plants, which lack Drosha homologues,
pri- and pre-miRNA processed by Dicer
homologue called Dicer Like(DL1) probably
takes place in the nucleus.
• Before miRNA duplexes transported to
cytoplasm 3, overhangs are methylated by a
RNA methyl transferase protien called
HEN1, then transported to cytoplasm by a
protien called HASTY(HST).
23

24. Dicer-like proteins:
 Class of RNase III endoribonucleases with two RNase III domains
and a PAZ (Piwi Argonaut and Zwille) domain.
The PAZ domain
is an RNA-binding module found
in Dicer-like proteins as well
as Argonaute (Ago) proteins.
24

25. RISC (RNA-induced silencing complex):
• RISC is a cytosolic complex with the Argonaute protein as a slicer.
• The 3D structure reveals a Piwi Argonaute Zwille (PAZ) domain that forms a
groove along with N-terminal, PIWI domain and middle domain.
• 30-end of small RNA binds to one end of PAZ cleft and the 50-end binds
near the other end of the PAZ cleft.
• The mRNA enters in the groove between the N-terminal and PAZ domains,
and comes out between the PAZ and middle domain.
• Since the groove is narrower on middle
domain side, the 50-end small RNA and
30-end mRNA on this side are closer
and paired by nucleotides, which will
bind mRNA to the whole RISC complex
25

26. • vedio
26

27. Mechanism of miRNA action
The degree of miRNA- mRNA complementarity
– key determinant of mechanism used
miRNA-directed mRNA cleavage
miRNA-directed translational repression
27

28. mRNA cleavage mechanism
• Most plants have near or perfect complimentarity,
mRNA cleavage.
• miRNAs guide , AGO component of RISC to cleave a
single phosphodiester bond of the target mRNA within
the miRNA-binding site.
• Fragments released and cleaved.
28

29. #
miRNA-directed translational repression
• Central mismatches trigger translational
repression
Example- miR172 in Arabidopsis ,regulates
flowering time and floral organ indentity

30. #
Summary of differences between plant and animal
miRNA systems
Plants Animals
miRNA genes: 100-200 100-500
miRNA biosynthesis: Dicer-like Drosha, Dicer
Mechanism of repression mRNA cleavage Translational repression
miRNA binding
sites in a target gene: Generally one Generally multiple
Functions of known
target genes: Regulatory genes Regulatory genes—crucial
crucial for development, for development, structural
enzymes proteins, enzymes

31. #
Target identification
• The duplex is partitioned into two parts, the seed part and the
out-seed part.
• 6-8 nt at the 5’ end of the mature miRNA sequence are very
important in the selection of target site.

32. #
Regulation of micro RNA gene transcription
The promoter regions of autonomously expressed microRNA (miRNA) genes are
highly similar to those of protein-coding genes
Activators and repressors of miRNA transcription
• Many TFs regulate miRNA expression positively or negatively in a tissue-
specific or developmental-specific manner.
• Eg:
(1) MYC and MYCN-
miR-17-92
(inhibit expression of
several tumour suppressor)
(2) p53 stimulates the
expression of miR-34 and
miR-107 families,
(which enhances cell cycle
arrest)

33. #
Regulation of miRNA decay
• Slow turnover is unlikely to be a
universal feature of miRNAs as they
often play a role in developmental
transitions or act as on and off switches,
conditions that require more active
metabolism.
• Several examples of accelerated or
regulated miRNA turnover are now
known. miR-29b decays faster in cycling
mammalian cells than in cells arrested
mitosis

34. Current methodologies for extracting
miRNAs
Silica-based miRNA recovery methods:
• miRVanaTM PARISTM
• and miRNeasy
Other kits
• MicroRNA Extraction Kit
• Sigma-Aldrich markets the mir Premier
microRNA Isolation Kit
widely used mini kits

35. Global quantity and quality assessment of miRNAs
• Several methods exist to determine the concentration and
quality of purified miRNAs. Spectrophotometric analysis is
one of the easiest and most common methods
• When the miRNA amount and concentration are sufficient,
quality assessment of the preparation can be performed by
capillary electrophoresis using the Small RNA kit in the
Agilent Bioanalyzer.
miRNAs profiling methods
(i ) qRT-PCR,
(ii ) microarrays,
(iii) Sequence specific hybridization in solution followed by
miRNA molecules counting based on reporter probes and
(iv) direct sequencing
35

36. Functions of miRNA
• Involved in the post-transcriptional regulation of gene
expression.
• Important in development.
• Metabolic regulation (miR-375 & insulin secretion).
• Multiple genomic loci (different expression patterns).
36

37. mi RNA editing
• Site-selective modification of RNA molecules at post-
transcriptional level to yield a product differing from the DNA
template.
• miRNA editing may represent a fine tuning process in miRNA
biogenesis at different steps, resulting in variations of target
mRNAs and providing another layer of regulatory controls
within the complex network of RNA-mediated gene functions.
• Catalyzed by the adenosine deaminases actingon RNA
(ADARs).
37

38. Edited miRNAs may silence a set of genes different from
those targeted by the unedited miRNAs, extending the
functional scope of miRNAs but increasing the complexity of
analyses at the same time.
38

39. A Peep into the Plant miRNA World
39

40. siRNA (small interfering RNA)
• Small interfering RNA (siRNA), known as short
interfering RNA, are a class of 20-25 nucleotide-
long RNA molecules that interfere with the
expression of genes
• They are naturally produced as part of the RNA
interference (RNAi) pathway by the enzyme
Dicer
http://en.wikipedia.org/wiki/Small interfering RNA
40

41. Mechanism of gene regulation by siRNA
41

42. • siRNAs bind to a ribonuclease complex called
RNA-induced silencing complex (RISC) that
guides the small dsRNAs to its homologous
mRNA target
• Consequently, RISC cuts the mRNA
approximately in the middle of the region paired
with the antisense siRNA, after which the
mRNA is further degraded
http://www.qbiogene.com/products/transfection/
app-sirna.shtml
42

43. What is the Difference between miRNA and
siRNA?
• Function of both species is regulation of gene expression.
• Difference is in where they originate siRNA originates with
dsRNA siRNA is most commonly a response to foreign RNA
(usually viral).
• Is often 100% complementary to the target miRNA originates with
ssRNA that forms a hairpin secondary structure.
• miRNA regulates post-transcriptional gene expression and is often
not 100% complementary to the target.
43

44. siRNA and miRNA si RNA mediated
degradation of
mRNA
versus
mi RNA mediated
inhibition of
mRNA translation
Filipowicz, Curr. Op. Structural Biology 15: 331-341 (2005)
44

45. siRNA miRNA
45

46. 46

47.  miR164 targets 6 OMTN, which are typical transcript factors and
respond to abiotic stress.
 Drought at the reproductive stage and over-expression of
OMTN2, OMTN3, OMTN4 and OMTN6 decrease the drought
tolerance of rice.
 Genes that are involved in stress, development, and metabolism
are down-regulated in OMTN transgenic lines during drought
stress, which have the opposite expression pattern in the wild
type during drought stress, indicating that miR164 negatively
regulates OMTN during drought stress.
 To identify the drought responsiveness of miRNAs in CWR shoot
and root tissues to provide a molecular understand for the
response of rice to drought. 47

48. Materials and Methods
O. Rufipogon  3-week-old seedlings drought stress
(16% PEG6000 for 24h)
 Small RNA isolation and sequencing (PEG6000-free and PEG6000-
treated groups)
Total RNA extracted by RNA extraction Kit method.
RNA libraries were constructed using a small RNA Sample Pre Kit
(constructed four small RNA libraries, called CL and CR from
PEG6000-freesamples and DL and DR from PEG6000-treated
samples).
The prepared libraries were sequenced using an Illumina HiSeq
2000 platform.
48

49. Data analysis:
• Removal of low quality reads, unknown base N, reads < 18 nt
and >30 nt in length
final clean reads (18±30 nt)
• Sequences matching the rRNA, tRNA, snRNA, and snoRNA
and repeats of the sequence tags were removed
The distribution of different small RNAs in four libraries. 49

50. Differentially expressed miRNAs in the root and shoot in response to
drought stress
• According to the biological characteristics of miRNAs,
novel miRNAs can be predicted by miRDeep2 software.
• Compared with miRBase.
• After getting the miRNAs, the expression level was
compared between the shoot (CL and DL) and root tissue
(CR and DR). A total of 200 miRNAs were differentially
expressed under 2 groups
50

51. 03/11/2018 51
A.The number of miRNAs
up- or downregulated
by drought treatment
B.A Venn diagrams
showing the unique
and shared miRNA
in the CWR root
and shoot under
drought stress
C.Hierarchical cluster
analysis of 31 miRNAs that
are regulated in both the
shoot and root. The fold-
change ratios of the miRNAs
are indicated by the different
colors.
Besides the known miRNAs, they also identified
97 novel miRNAs. All of the novel miRNAs were
equally distributed in the 12 chromosomes of the
rice genome, except orumiR76 and oru-miR97,
which were unknown

52. The distribution of novel miRNAs in
chromosomes.
52

53. qRT-PCR validation of the drought responsiveness
of miRNAs
• Chosen 12 differentially expressed miRNAs that were
expressed in response to drought from the shoot and
root (2 novel miRNAs, oru-miR21 and oru-miR47 +2
known miRNAs (miR171f and miR395f) to insure the
accuracy of qRT-PCR.
• First, miR171f and miR395f expressed in the root:
miR171f was up-regulated, and miR395f was down-
regulated .
• The results are consistent with reports and sequencing
data, thus indicating the accuracy of the qRT-PCR.
53

54. qRT-PCR validation of drought-responsive miRNAs in shoot and root.
The expression values presented are the means of
three technical replicates. OsActin was used as the reference gene.
54

55. • At the miRNA level, 77 known miRNAs, which belong to 23
families, including 40 up-regulated and 37 down-regulated in
the shoot, and 85 known miRNAs in 46 families, including 65
up-regulated and 20 down-regulated in the root, were
identified as differentially expressed.
• In addition, predicted 26 new miRNA candidates from the
shoot and 43 from the root that were differentially expressed
during the drought stress.
• The differentially expressed miRNAs that are tissue-specific
under drought conditions could play different roles in the
regulation of the auxin pathway, the flowering pathway, the
drought pathway, and lateral root formation.
55

56. w There are no previous reports on miRNA responses to
nematode infection in cotton.
w In this study, selected 28 plant growth and nematode-related
miRNAs and investigated their miRNA and target gene
responses to RKN infection in cotton.
w Cotton (Gossypium hirsutum L.) cultivar ‘Texas Marker 1
(TM-1)
w After one week of germination, the cotton seedlings were
divided into two groups: one for controls and another to be
infected with RKN.
56

57. Methods
 RNA extraction: mirVana™ miRNA Isolation Kits
 miRNA-target gene expression analysis:
Expression of these selected miRNAs and their target genes
was determined by quantitative real-time PCR (qRT-PCR)
using an ABI 7300 Sequence Detection System SYBR Green
ROX™ qPCR Mastermix was used to determine the
expression levels of miRNAs and their target genes
 During gene expression analysis, UBQ7 and actin were
served as reference genes. The UBQ7 and actin are
housekeeping genes
57

58. Total (=28 miRNAs) : gene expression analysis
• 14 miRNAs which responded to plant-
parasitic nematode infection in other
plant species
• These 14 miRNAs were miR167,
miR390, miR396, miR2950, miR7502,
miR7504a, miR7504b, miR159,
miR397, miR398, miR408, miR827,
miR862 and miR2119.
• The second group
included miRNAs that
play important roles in
plant growth or
development,
including root
development
Previous reports have identified few miRNA targets in cotton
These 8 miRNA-target pair were miR167-ARF8, miR396-GRF1, miR319-
TCP4, miR159-MYB, miR408-LAC (laccase-4-like), miR156-SPL1,
miR156-SPL9 an miR398-SOD (copper/zinc superoxide dismutase).
58

59. 59

60. The expression levels of 8 tested miRNA target genes in cotton roots under normal
growth condition without RKN infection. The expression levels are compared with
the expression average of two reference genes which is normalized to the value of 1
for y-axis.
60

61. Root knot nematode infection affected miRNA expression in cotton roots
after 10 days of infection. The y-axis shows the expression fold changes
after infection. 61

62. Root knot nematode infection affected the expression of
miRNA targets in cotton roots after 10 days of infection. The y-
axis shows the expression fold changes after infection.
62

63. Relative expression of miRNAs and their targets in cotton roots under root knot nematode
treatment and untreated controls. The figures show that negative or positive relationship
between the expression of miRNAs and their corresponded targets.
63

64. In this study, found that several of the tested
miRNAs are responsive to RKN infection showing
expression changes from down-regulation by 33% to
up-regulation by 5 fold following 10 days of
exposure with RKN.
Among these miRNAs, miR319 was induced by
500% by RKN and it is the most responsive miRNA
in this study, which is accompanied by its targeted
inhibition.
The miR319 regulates plant development by
controlling TCP (transcription factor )family.
The TCP transcriptional factor family has been
found to regulate various plant developmental
processes.
Thereby the manipulation of miR319/TCP and
miR159/MYB regulatory modules to combat RKN
infection in cotton warrants future investigation. 64

65. • Potato anthocyanin accumulation was extensively studied in
different cultivars previously.
• Potato tubers are potent sources of antioxidants and some
studies reported that tubers are rich in polyphenols.
• Additionally, these bioactive compounds have potential health
benefits including anticancer and immunomodulatory
activities.
• miR828 is able to direct cleavage of the RNA originating from
Trans-acting siRNA gene 4 (TAS4)
65

66. Number of potato cultivars were chosen for studying the role of
miR828 having contrasting tuber skin and flesh colours
66

67. RNA gel blot analysis of mir828.

68. 68
Expression analysis of MYB genes in cultivars. In tuber skin and flesh samples
qPCR was used to analyse the expression of MYB73-like and MYB12

69. Regulatory roles of miRNAs in plant
development
Phase transition
 developmental phase transitions - seed
germination, vegetative phase change,
reproductive phase change, flowering initiation,
seed production
 miR156a gene causes late flowering and delays
vegetative phase change
69

70.  In Arabidopsis early activation tagged (eat-D) mutant
exhibits early flowering with disrupted floral
structure(miR172)
 Mutation in ago –altered juvenile to adult vegetative to
reproductive phase change
70

71. Organ development
 Leaf morphogenesis(miR-JAW in Arabidopsis,
CIN gene in Antirrhinum majus)
 Identity of floral organs and flowering
time(miR172 in Arabidopsis)
71

72.  Organ boundary and organ polarity(miR164)
 Root development
miR160 regulate lateral and adventitious root
formation via auxin signaling
 SAM development(Shoot Apical Meristem)
miR165/166 plays a primary role in meristem
formation
72

73. Vascular development
• miR165/166 and its targets play an important
role in vascular development
Stress responses
• miR398- maintains the copper homeostasis by
regulating CSD1 and CSD2 through mRNA cleavage
• miR395 regulates sulphate assimilation
Growth hormone signaling
• miR160 and miR167 regulate the ARF genes in auxin
signaling
73

74. MicroRNA Targets Prediction
Databases
• Most of the computational tools developed to identify mRNA target
sequences depend heavily on complementarity between miRNA seed
sequence and the target sequence
• Most methods mainly use sequence complementarities,
thermodynamic stability calculations and evolutionary conservation
among species to determine the likelihood of formation of a productive
miRNA-mRNA duplex.
• Incorporating mRNA UTR structure to predict microRNA targets
(Robins et al. 2005)
– Make sure the predicted target is “accessible”.
– Not forming base pairing itself.
74

75. miRNA
Tools
75

76. miRNA
Tools
FEBS Journal 276 (2009) 2150–2156
76

77. 77

78. http://diana.cslab.ece.ntua.gr/?sec=home
78

79. 79

80. CONCLUSION
The RNA silencing has practical use because of the ability to reduce
gene expression in a manner that is highly sequence specific as well
as technologically facile and economical
However the major obstacles hindering its immediate applications
RNA Interference: Its Concept and Application in Crop Plants
include selection of targeting sequences and in the delivery of
siRNA. The key issues are
(1) how to select silencing targets for a particular disease and
(2) how to efficiently deliver siRNAs into specific cell types in vivo?
However a better and comprehensive understanding of RNAi should
allow future plant researchers to work effectively and efficiently in
order to improve crop plants nutritionally and manage various
mascotous intruders of crop plants.

81. #

82. ???...........
82

83. 83

84. • Cold-regulated gene expression is important for plant
tolerance to chilling stress and cold acclimation.
• Cold response involves different metabolic pathways,
gene regulation pathways and cell compartments.
• Understanding of the regulatory mechanisms in
responding to cold stress is therefore important in
genetic engineering of plants to improve tolerance to
low temperature stresses.
84

85. • 3 TFs -CBFs (CRT/DRE binding factor) or DREBs (DRE
binding protein) can bind to DRE/CRT (dehydration-
responsive element/C-repeat) in the promoters and activate
transcription of the COR(cold regulated)/KIN (cold
induced)/LTI(low temperature induced)/RD (responsive to
dehydration) genes.
• The CBF genes are transiently induced by low temperature,
and this induction precedes that of COR/KIN/LTI/RD genes.
• CBF1 or CBF3 in Arabidopsis results in constitutive
expression of downstream cold-inducible genes, high levels of
proline and soluble sugars, and increased freezing tolerance.
• CBF2 negatively regulates the expression of CBF1 and CBF3
in the cold, and the CBF1/DREB1B and CBF3/DREB1A
positively regulate cold acclimation by activating
CBF/DREB1-target genes.
85

86. Generation of Transgenic Arabidopsis Plants Over-expressing
miR397a
Identification of Arabidopsis miR397a ,22 nt microRNAs located
on chromosome 4,
• A 300 bp fragment that includes the foldback structure of
miR397a was amplified from genomic DNA.
• The amplified fragments were digested with restriction
enzymes XbaI and KpnI and cloned downstream of the CaMV
35S promoter in a binary vector (pCAMBIA2305).
• The correct sequence was confirmed by sequencing of the
insert.
• The resulting construct was electroporated into Agrobacterium
tumefaciens GV3101, and to transform Arabidopsis thaliana.
• Examined by small RNA blot analysis and Compared to wild-
type plants.
• Four of them showed high levels of miR397 transcripts. 86

87. The sequences and predicted fold-back structures of
miR397a
• RNA blotting showing transcript levels of
miR397a precursor in
• miR397a over-expressing (miR397-ov)
plants. Wild-type (WT) and four
independent transgenic lines (labeled as
1, 2, 3 and 4) of miR397-
• ov plants were analyzed. A photo of the
ethidium bromide-stained tRNA is
included as a loading control.
87

88. miR397a-ov Transgenic Plants Displayed
Freezing Tolerance
88

89. RNA Blotting Analysis of Cold-regulated CBF and
Downstream Cold Responsive Genes in miR397a-ov
Transgenic Plants
89